DAY 3: Consumer Awareness: Product Safety and … · Web viewConsumer Awareness: Personal Care Products Safety and Labeling Lesson 3: Is it safe? Basic toxicology - Safety testing:

File: Lesson 3.doc NWABRAug. 2008

Consumer Awareness: Personal Care Products Safety and Labeling

Lesson 3: Is it safe? Basic toxicology - Safety testing: MSDS’s andanimal studies - Designing a human study

Summary: Class discussion about the homework followed by demonstrations, discussion about basic toxicology principles and dose-response worksheet. Small group activity with MSDSs. Class review of studies with animals and designing human studies.

Lesson Objectives:1. Students will be able to identify three basic principles of toxicology.2. Students will be introduced to material safety data sheets (MSDSs) as a source of cosmetic

ingredient safety information. 3. Students will review the steps in ingredient development and safety evaluation and be able to

explain the role of animals in safety testing.4. Students will gain an understanding of basic considerations required when designing a human study.

(who, what, when, where; how to obtain reliable results)

Materials & Preparation:Distribute sample commercial products/containers to lab group benches;

approximately 2-4 containers per group (the containers provided with "active ingredients" are not useful for this exercise)Copy S3.1, Dose-Response Worksheet, one for each studentCopy S3.2, Cosmetic Ingredients: Function and Toxicity Chart, one for each studentOptional: TG3.15b, 1 copy per group; cut apart and put activities in appropriate order Copy (or use from kit) four different MSDS’s, one set of 4 to each groupCreate overheads: TG3.1, Sense (or Cents) Behind Safety

TG3.3, Safety warning; reporting TG3.4, Toxicology definition TG3.5, Toxicology principles TG3.7, Dose TG3.7b, Hazard, risk TG3.10, Dose response curve TG3.13, Skin biology

TG3.14, Skin absorption TG3.15, Cosmetic ingredient development TG3.16, Drug development process TG3.17, MSDS purpose and availability TG3.21, Animal claims (2 pages) TG3.22, Animal studies – background TG3.24, IACUC and protocol content TG3.25, 3 R's of animal studies TG3.27, Human Study Example (3 pages) TG3.28, Lotion Study Basics

Copy S3.3, Winning Combination, one for each student (homework)

Engagement: Yesterday we made a lotion, today we are going to talk about whether it can be considered safe enough to put on the market.

Northwest Association for - 1 - Lesson 3 Biomedical Research


Has anyone ever had an allergic reaction or skin irritation from a cosmetic? If so, what did you do about it?

(Quit using the product; contacted manufacturer or FDA; avoided that product and ones with similar ingredients)

As you answered your homework questions you had a chance to think about both manufacturers’ and consumers’ responsibilities in keeping cosmetics safe.

(10 min.) Class discussion about homework assignment Overhead: “Sense (or Cents) Behind Safety” (S2.3 homework) OH TG3.1Teacher Guide with answers/discussion points TG3.2

It is important to remember that if no safety testing was done, the labelmust contain a warning. OH TG3.3The FDA encourages consumers to report problems with cosmetic products to them, as well as to the manufacturer.

Principles of Toxicology OH TG3.4The process of determining the safety of cosmetic ingredients falls under thescience of toxicology. Originally it was the science of identifying poisons and determining how much would kill you. Today, toxicologists are involved in identifying harmful effects, assessing the probability of harm, and determining safe levels of many agents on humans, animals and the environment.

You may have heard the statement: “The dose makes the poison.” Any substance or ingredient can be toxic depending on its amount or dose and individual sensitivity. Even too much water can dilute the body’s sodium levels and cause hyponatremia – a potentially series health problem.

There are three basic principles of toxicology that must be considered OH TG3.5when determining the safety of any compound, ingredient, or chemical:

Background OH TG3.61. Dose-response relationship2. Hazard vs. risk 3. Individual susceptibility

Dose-Response RelationshipIt is not hard to come up with examples of chemicals, foods, or environmentalpollutants that cause increasing harm (or effects) when present at increasingconcentration

Example: UV rays and sun tanning/burning Caffeine in coffee or coke and stimulation/jitters Air particulates and breathing/coughing

The term dose refers to the amount of a chemical that actually enters the body,through lungs (inhalation), mouth (ingestion), or skin (dermal absorption).

The actual dose depends on variations of exposure to the chemical: OH TG3.7Duration and combined exposure from multiple sourcesFrequency and time between exposuresBody size



Dose Determination for CosmeticsWe expect cosmetic manufacturers to determine the safety of their ingredients (and products) based on an expected “dose” (amount used per application) of the product. Determining the “dose” of cosmetics can be very difficult. The label doesn’t state a “dose” or “serving size” like drug or food labels.

Dilemmas for manufacturers planning safety tests:- The manufacturer expects customers to use the lotion on their hands, but they like it so

much they use it on their hands and legs. - The manufacturer plans to test the safety of use over one month, since that would be

equivalent to one container of the product, but consumers like the product so much that they use it continuously for years.

- Some of the ingredients in the manufacturer’s lotion are also found in two or three other products that consumers use everyday.

- If a product is expected to be used for years, does the manufacturer need to test for the potential to cause cancer, or birth defects if used by pregnant women?

Hazard vs. RiskHazard can be thought of as the potential for a chemical to be harmful, butit doesn’t mean that it will be harmful under all conditions. OH TG3.7b

Risk is the likelihood (or probability) that you will be hurt or harmed.Determining your risk means taking into consideration the

inherent hazard level of the chemical,the dose-response relationship,and all aspects of exposure (frequency, duration, body size).

Hazard is not synonymous with risk.

Individual SusceptibilityIndividual susceptibility refers to differences in sensitivity to toxic agents.

For some people a bee sting is a nuisance, for others it can be deadly.For some people peanuts are a great snack, for others they can be deadly.

Susceptibility is influenced by age, gender, health, genetic background and life style. Sensitivity can develop after repeated exposure. Hair dyes tell youto do a skin test before every use, even if you haven’t had a problem in the past.

[These basic principles are explained well in the power point slides from UW TG3.8Center for Ecogenetics & Environmental Health, Community Outreach & Education Programs. The slides refer to chemicals in the environment, but the information applies to cosmetics.]

Demo Dose-Response Demo TG3.9 pg 1For most people, the greater the dose of a substance, the greater the response.This can be seen in a simple demonstration with glasses of water, representing water in the body or the bloodstream, or with petri dishes representing surface area of the skin. The more lotion you put on, the more of the ingredients you are exposed to. If yourskin is sensitive to an ingredient or chemical, the greater the dose, the greater your reaction to it.

TG3.9 pg 2



But size and weight can also effect the response to a given dose. The amount of caffeine in one can of cola may have little effect on an adult, but that same can of cola can have a greater effect on a child because of less body weight or blood volume.Size and weight also effects response to cosmetic ingredients. If an adult and child use the same amount of lotion (for example, 1 tablespoon), it may cover a greaterproportion of skin surface area of the child and ingredients that penetrate will be in greater proportion to the water/blood volume of the child.

Dose Response CurvesScientists record the experimental response to different doses of a TG3.10chemical in a dose-response curve. The x-axis is the dose, measured (see R3.2)in mg/kg, milligrams of chemical per killigram of body weight.

The y-axis is the percent of subjects showing a response which may be death or, for cosmetics, skin reaction (redness, inflammation, blistering).

Knowing the dose-response of a chemical is essential for knowing its cause and effect profile. A comparison of dose-response curves allows one to compare one chemical to another. Three important doses are determined from the curve. (see TG3.10)The shape (slope) of the curve reflects potency. The more vertical the slope, the greater the chance of “overdosing” with small changes in dose.

Demo Variable Response to Dose TG3.11All people don’t respond to all chemicals in the same way.Bee stings and peanuts are tolerated by most, but are deadly to a few.

Teacher or students can demonstrate variability to response.Discuss factors that are responsible for variable responses.(genetics, age, size, health, medications, life style)

Worksheet Dose-Response Worksheet: How much is too much?You now have the opportunity to generate a dose-response curve and evaluate several dose-response curves based on what we have just talked about. S3.1

On the first page you will read about a hypothetical human safety test of a new lotion. You will be asked to graph the results that are described and then answer somequestions about the graph and the information you learn from such a graph.

On page 2, you are given the results of toxicity tests for two new lotion ingredients.You are then asked some questions about the differences in toxicity between these two ingredients.

Answer key TG3.12Students may want to measure and see how much lotion represents1 mg/kg in an average 70 kg human.

Skin FunctionDetermining the amount of a cosmetic ingredient that gets into or through the skin and into the blood circulation is important in assessing the ingredients’ safety.Discuss biology of the skin – OH TG3.13

Function and layers of the skin (see R3.3)



Normal skin pH is somewhat acidic – in the range of 4.2 to 5.6, varying from one part of the body to another. The acidity inhibits the growth of foreign bacteria and fungi.

What are the routes of entry for chemicals on the skin? OH TG3.14Chemicals must reach blood vessels within the dermis to be spread Metabolism – breakdown within skin cells can change the chemical

Ingredient Evaluation ProcessThe process of evaluating the safety and efficacy (function) of new ingredients for cosmetics and medicines makes use of the principles of toxicology within a hierarchy of research and development steps. OH TG3.15

Optional: Make copies of TG3.15b, cut apart, and ask students to put them in appropriateorder before showing TG3.15 TG3.15b

Drug development and safety testing is a more structured process based on regulations and FDA approvals. It includes many of the same initial stepsas in development of new cosmetic ingredients however, there are Food & DrugAdministration (FDA) submission and approval steps and the human testingstep (called clinical trials) is longer and more involved. OH TG3.16

The results of tests “in vitro”, in cell culture, and in animals must bepresented to the FDA in an “investigative new drug” (IND) application. The INDalso includes information about the manufacturing process, label (for pre-approvalby FDA), and the plan for clinical trials (studies in humans).

(See R3.4, R3.5)After a drug is approved, most of the information is available to the public through the “Freedom of Information” Act. Since cosmetic ingredient safety data is not submitted to the FDA, it is harder for a consumer to find.

Introduction to Material Safety Data Sheets (MSDS) Prompt: There is a way for the public to get some information about the safety of cosmetic

ingredients, and specifically, find animal toxicity data. That is what we are going to explore now through Material Safety Data Sheets (MSDS’s).

Group Activity: Divide students into groups of 4 students. Each group should have:1) 3-4 sample products/containers from the kit

Hand-out 2) “Cosmetic Ingredients: Function and Toxicity Chart” (one per student) S3.2Hand-outs 3) Four different MSDS sheets Binder – Back pocket

Option 1 – Each group has 4 different MSDSs but all groups have the same 4 MSDSs.

2 MSDSs will correspond to common ingredients found in cosmetics(propylene glycol, methyl paraben)

2 MSDSs will be for an ingredient in the lotion made in class (borax, mineral oil)

Option 2 – From the web site, copy a different MSDS for each student.NOTE: MSDSs from different companies are similar in format but not identical in content. Not all MSDSs will contain animal toxicity data.



Teacher will lead the class discussion.Each of you within a group has a Material Safety Data Sheet (MSDS) for a different ingredient. As you look at the information in your MSDS, whowould you say could use this information?

Overhead: Purpose of MSDS’s and public availability OH TG3.17Discussion points TG3.18

Information found in an MSDSFocus on synonyms Focus on section 11, “Toxicological Information”

Animal testing, routes of delivery, meaning of LD50

Discussion points TG3.19Optional information: mouse LD50 converted to human LD50 TG3.20

Group activity: S3.2a) Complete the “Cosmetic Ingredients: Function and Toxicity Chart” by using the MSDSs to identify what type of animal tests have been done.b) Study labels on the sample products/containers and determine if any of

those ingredients are found in products labeled “not tested in animals”. OH TG3.21 pg 1

Prompt: There are many claims that we can’t check because we don’t have access to testing data or there is no standard or definition for some claims. As you have discovered, we CAN determine whether some ingredients have been tested in animals. Animal-tested ingredients include those used in our class lotion and in manycommercial products.

Several days ago, we talked about labels that claim “not tested in animals”. Today we find that the ingredients have been tested in animals. How can companies make that claim? OH TG3.21 pg 2

(ingredients were tested but not the final lotion; someone else did the testing; tests were done greater than 5 yrs ago)

Animal StudiesBrief class discussion:

Overhead: Animal studies - Background OH TG3.22Discussion points (also see articles under “Research with Animals”) TG3.23

Although no animal is a perfect imitation of humans, animal models allow us tojudge the effects of multiple cellular interactions and metabolism.

Safety tests on new ingredients for cosmetics and drugs usually include animalstudies. There are no adequate non-animal tests to replace all animal tests but many previously used animal tests have been replaced.

Safety testing is done for a variety of reasons: To find the safe dosing range, to determine the potential for skin irritation, allergy, or potential for harm if the compound gets through the skin and into the blood stream.

These end-points are hard to achieve in non-animal models.

Cosmetic ingredients that have previously been tested or have a history of safe use don’t have to be re-tested on animals, although they may require human testing.



When testing is required on new compounds, animal studies are done OH TG3.15after gathering data from non-animal tests. The use of animals in researchis not taken lightly. Federal laws govern the care, research, housing, & transport and unannounced inspections by US Department of Agriculture are mandated. By law, all animal study protocols must be approved in advance.

Role of IACUC TG3.24

When animal studies are needed, the guiding principles are the 3 R’s Overhead: replacement, reduction, refinement OH TG3.25Discussion points and examples TG3.26

To replace an animal test with a new test requires “validation” and approval by government agencies. The protocols must be repeatable by multiple labsand the results must be reproducible.

See R3.6 for description of common safety tests done on cosmetic and drug ingredients. R3.6

See R3.7 – a New York Times article on the development of alternativesto animal testing because of European regulations R3.7(Also see “Protecting More than Animals” in Research with Animals and curriculumsummary for “For the Greater Good” in NWABR Curricula

Human Lotion Studies

If ingredients appear safe in in vitro tests and animal studies, the next studies are conducted in humans. Designing a human safety study, or a lotion quality study, is like designing any scientific investigation. Here is an example of a hypothetical human lotion study. OH TG3.27

What do you think about this description? Does it meet the criteria for being informative, reliable, and reproducible?

The design of a human lotion study must incorporate all of the informationrequired in a science lab report. OH TG3.27 p 2The hypothetical study description touches on essential aspects of a humanstudy, but it lacks procedural details which makes it non-reproducible. OH TG3.27 p 3

An adequate “procedure” must include details:who, what, when, where, purpose, and how the evaluation will be done.

Within these categories, there are many ways that a human lotion study couldbe designed, depending on the purpose(s) of the study.

You should be able to identify the controlled, manipulated, and respondingvariables in the study design.

Group Working in small groups, students should discuss possible study designActivity: options under each category of Lotion Study Basics. OH TG3.28

Class discussion led by the teacher: Have each group report on one of their study design categories.

Discussion points TG3.29



Closure: Over the last three lessons you have had an opportunity to investigate cosmetics on many levels. - You should have a better understanding of cosmetic regulations. - You should be more aware of what constitutes puffery vs fact when you read those claims on cosmetic containers. - You know where to find safety information about cosmetic ingredients.- You have a basic appreciation of toxicology principles and the development andsafety evaluation process for cosmetics.

The finale of the unit is to put yourself in the role of the product safety manager, or toxicologist. You must consider whether animal studies are needed, then design a human lotion study and the finished label for your lotion.

There are many ways to design a human lotion study but it takes good planning to design a study that produces results that are reliable and reproducible. If consumers complain to FDA about your product, the FDA can ask to see your safety test results. If they feel that you didn’t adequately test the product, then you can be forced to pull it off the market if a court agrees with the FDA.

Homework Assignment: Your homework assignment is to a) assess the need for animal studies on your lotion, b) using the Lotion Study Basics as a guide, design a human safety or quality study for your lotion, c) assume that the lotion passes your safety test; prepare a final label for your lotion, including claims and other required parts of a label.

“Winning Combination” S3.3Teacher guide: "Winning Combination" evaluation rubric TG3.30

Resources: (yellow pages)

FDA: Animal Testing FDA websiteLesson 3 Website resources R3.1Dose-response background R3.2Skin layers R3.3Drug development process R3.4Drug development process – 2 R3.5Safety and quality tests R3.6Animal alternatives NYTimes R3.7


File: Sense safety - OH.doc NWABR Feb. 2005

Northwest Association for Teacher Guide (TG)3.1 Biomedical Research. Overhead

File: Sense safety - OH.doc NWABR Feb. 2005

Sense (or Cents) Behind Safety

1. Why would a cosmetic company perform safety tests?

2. What do you expect cosmetic manufacturers to do to make products safe?

3. What are the consumer’s responsibilities for keeping cosmetics safe to use? (What does industry expect consumers to do?)

4. If you were a cosmetic manufacturer, what safety tests would you do before your product went on the market?


File: Sense safety - TG.doc NWABR Feb. 2005

Northwest Association for Teacher Guide (TG)3.2 Biomedical Research.

File: Sense safety - TG.doc NWABR Feb. 2005

Sense (or Cents) Behind Safety

1. Why would a cosmetic company perform safety tests?

Law requires a product to be safe - but FDA doesn’t review safety test results before marketing

Company’s reputation for safe products would be at risk if there were a problemTo avoid legal suits (and monetary fines) by consumers that might be hurt by the product

2. What do you expect cosmetic manufacturers to do to make products safe?

Perform appropriate safety testingInclude cautions/warnings on the labelsProvide directions for useUse only high quality, non-adulterated ingredients

Accurately label the products so consumers can avoid ingredients that may cause them allergic reactions.

NOTE: There is no way to guarantee that products will be non-irritating or non-allergenic to all people.

3. What are the consumer’s responsibilities for keeping cosmetics safe to use?(What does industry expect consumers to do?)

Don’t share – to prevent contamination and spreading bacteriaPrevent bacterial contamination Keep containers closed and out of sunlight Discard products if they are discoloredDon’t add water, or saliva, to dried products

4. If you were a cosmetic lotion manufacturer, what safety tests would you do before your lotion went on the market?

New ingredients require more extensive testing, including animal tests.Students may say no animal tests are necessary since all of the individual ingredients are safe and previously tested.

Types of tests that could be done:Skin irritation, eye irritation, effects of oral ingestion, allergy tests

Northwest Association for Teacher Guide (TG)3.2 Biomedical Research.

File: Safety warning.doc NWABRJan. 2006

Who is responsible for safety testing?

Cosmetic manufacturers

Cosmetic Ingredient Review (CIR) panel = industry safety committee

FDA Requirement:

If a cosmetic product has not been safety tested, FDA requiresthat a warning be on the label.

WARNING: The safety of this product has not been determined.

FDA Request:

FDA encourages consumers to report problems with cosmetic products to them, as well as to the manufacturer.

1. Call your local FDA officeSeattle area - Consumer complaints: 206-553-7001

2. Write: FDA Center for Food Safety and Applied NutritionOffice of Cosmetics and Colors200 C St., S.W.Washington, DC 20204202-401-9725

Call: CFSAN Adverse Event Reporting System (CAERS)301-436-2405

Email: [email protected]

3. Contact the CFSAN outreach and information center:1-888-723-3366


mailto:[email protected]

File: Toxicology intro.doc NWABRJan. 2008

Toxicology

Old Definition:The study of poisons.

How much will kill you?

Newer Version:

The study of the harmful affects of chemical or physical agents on living organisms (humans, animals,and the environment).

Northwest Association for Teacher Guide (TG)3.4Biomedical Research. Overhead

File: Tox principles.doc NWABRJan. 2008

In order to investigate the safety of a new cosmetic ingredient, one must consider the

Three Principles of Toxicology:

1. Dose-Response Relationship

2. Hazard vs. Risk

3. Individual Susceptibility


File: Tox principlesbkgd.docNWABR

Jan. 2008

Principles of Toxicology

1. Dose-Response Relationship: The larger the dose, the more extreme the response will be.

Dose: Amount of hazard that comes in contact with your body

Exposure: Contact with your body can come from various routes (skin, lungs, swallowing)

Safety, or harm, from exposure depends on:DurationFrequency Time between exposuresCombined exposure from multiple sourcesBody size

Response: Reaction to the dose;Can range from no effect to death

2. Hazard vs. Risk

Hazard = Anything in the environment that could harm you.Assessment may be based on scientific studies or

personal experience; Harm may not be immediately obvious

Risk = The likelihood of harm in defined circumstances. Assessment based on exposure and degree of hazard

Hazard is not synonymous with Risk

3. Individual Susceptibility: differences in sensitivity to toxic agents

Age, gender, health, genetic background, life styleSensitivity can develop after repeated exposure

Reference: Gilbert, Steven G. “A Small Dose of Toxicology”. 2004, CRC Press LLC.www.asmalldoseof.org

Northwest Association for Teacher Guide (TG)3.6Biomedical Research.

http://www.asmalldoseof.org/

File: Tox principlesbkgd.docNWABR

Jan. 2008


File: Dose.docNWABR

Jan. 2008

Dose can depend on…

http://whs.wsd.wednet.edu/Faculty/Young/YNHC/ynhc.htm Teacher Guide (TG)3.7Overhead

Duration of Exposure:How long?

S S

M L

Frequency of Exposure: How often?

Body Size:How big or

small are you? you

File: Hazard, risk.doc NWABRJan. 2008

http://whs.wsd.wednet.edu/Faculty/Young/YNHC/ynhc.htm Teacher Guide (TG)3.7bOverhead

File: Hazard, risk.doc NWABRJan. 2008

Hazard is not synonymous with risk

Hazards could harm you. Bacteria & Viruses

Harmful Chemicals

Tobacco & Smoke

Stress

Loud noises

http://whs.wsd.wednet.edu/Faculty/Young/YNHC/ynhc.htm Teacher Guide (TG)3.7bOverhead

Risk is the likelihood of harm in defined circumstances

File: Dose-response demo.doc NWABROct. 2006

Demonstrating the Principles of Dose-Response

Adapted from: “A Small Dose of Toxicology”, by Steven G. Gilbert. 2004, CRC Press LLC.www.asmalldoseof.org

Demonstrating the Importance of the Amount of the Dose:The greater the dose, the greater the effect

Materials:Three large size glassesThree petri dishesFood color (blue is best)One pitcher of water

Add approximately equal amounts of water to three glasses. This represents the approximate water content of an individual.

To visualize skin surface area when thinking of doses of cosmetic ingredients, add equal, small amounts of water (to cover the bottom) to three petri dishes

Put one drop of blue food color in the first container (glass or petri dish), three in the second container and then 6-9 in the last container.

Stir with a pencil or pen and discuss the change in color as a response to increased dose of food color in each glass. Discuss how some chemicals, caffeine being one, distribute throughout the total body water.

Some chemicals from cosmetics can penetrate through the skin and can be distributed through the blood stream while others remain on or near the surface of the skin.

Dose / Response - A Demonstration 5/25/04A Small Dose of Toxicology

Effects of Amount on Response




Demonstrating the Principles of Dose-Response

Adapted from: “A Small Dose of Toxicology”, by Steven G. Gilbert. 2004. CRC Presswww.asmalldoseof.org

Demonstrating the Importance of SizeThe smaller the size, the greater the effect

Materials:One large and one small size glassOne large and one small petri dishFood color (blue is best)One pitcher of water

Fill the large and small glass (and the large and small petri dish) to approximately ¾ -full with water. The small containers represents a small child in contrast to the adult size containers.

Put one drop of food color in each container. The small glass will be much darker and usually look like the high dose glass from the first demonstration.

Discuss the importance of size, and the impact weight has on dose, depending on sophistication of the group. A small child that drinks one can of caffeinated soda will have a very different response than an adult because of the difference in the dose of caffeine relative to body size.

Dose / Response - A Demonstration 5/25/04A Small Dose of Toxicology

Effects of Size on Response





File: Dose-response curve.doc NWABRJan. 2008

Dose-Response Curve:

Principles of Toxicology – 4/12/04A Small Dose of Toxicology

Dose (mg/kg body weight)Increasing dose

Dose Response Function

0

25

50

75

100

0 10 20 30 40 50 60 70 80 90 100

% R

espo

nse

NO Effect

All Effected

Half Effected

www.asmalldoseof.org

X axis: Dose = expressed in mg/kg.mg = milligrams of substance/chemicalkg = killigrams of body weight

Y axis: Response = % or number of subjects with pre-defined end-point (inflammation, death, etc.)

Important information from dose-response curves

Dose at which: no effect is seen50% of subjects have the “response”see maximum response

Shape of curve reflects potency



File: Variable response demo.doc NWABRJan. 2008

Variability in Response Demonstration

Adapted from: Tox-in-a Box, Community Outreach and Education ProgramNIEHS CEEH, University of Washington; http://depts.washington.edu/hereuw

Materials and EquipmentLab coat, safety glasses, and glovesDistilled water (approximately 400 ml)Bench cover50 cc tube containing 35-350 mg bromophenol blue free acid (BPB) or

15 cc tube containing 17-25 mg BPB0.1 N NaOH (20 ml)0.3 N HCl (approximately 3 drops)Spoon for stirringPipettes4-5 clear plastic cupsBlank sheet of white paper

Premise:The dye/buffer solution will resist color change until the buffer capacity is exceeded. After

the capacity is exceeded, the solution will change color (from blue to yellow) rather quickly with each additional drop due to the pH sensitivity of BPB.

Preparation:Note: THE BPB solution is a dye and will stain clothes, tables, etc. Bench covers should be used to protect the countertop.

1. For a large amount of BPB solution add 20 ml of 0.1 N NaOH to 35-50 mg BPB. For a smaller amount, add 10 ml of 0.1 N NaOH to 17-25 mg BPB.

Note: The BPB solution is not stable for more than a day or two; do not use tap water in this experiment, as it is often quite alkaline. Use only distilled water.

2. Fill four or five clear plastic cups with distilled water.

Experiment: Take four or five glasses each filled with distilled water, one of which as a few drops of 0.3 HCl. Then add three drops of BPB to each one. Stir each cup with the spoon provided.The cup with the HCl will respond differently (it will turn yellow) and represent the “sensitive” individual. Holding a blank sheet of white paper behind the cups helps the students see the color changes.

Some people may respond differently to the same dose of a chemical because of genetics, diet, or other factors. You can brainstorm with the students why some people are more sensitive or resistant than others to chemicals.

Clean Up:At these concentrations, these compounds will be similar to table salt and thus are not toxic and will not harm the environment. The waste, therefore, can be poured down the drain.


http://depts/

File: Lotion dose response key.doc NWABRAug. 2008

Dose-Response Worksheet: How much is too much?

Name __________________________ Period _________ Date __________

Hypothetical: Human Safety Test of a New Lotion

A local cosmetic manufacturer has developed a new moisturizing lotion. The lotion has passed all of the animal-based safety tests and is now ready for testing on humans. Healthy males and females have been recruited for the study. They have been fully informed about the protocol and the risks and have voluntarily signed informed consent forms.

Ten people are participating in the study. Each person will be given a pre-measured, single use packet of lotion to rub on their right, inside forearm. Each day for 20 days, they will come to the clinic and be given a new packet of lotion to apply to their right, inside forearm. A clinical research associate (CRA) will observe the arm and record skin appearance before application of the next packet of lotion.

Following is a summary of the results of the study based on the CRA’s observations. Please graph the results below, labeling the axes appropriately.

Day 0: All 10 arm surfaces looked normal and healthyDays 1-4: After 4 applications, all 10 arm surfaces look normal and healthyDay 5: After 5 applications, 1 person’s arm had turned green in the area of lotion applicationDay 7: After 7 applications, 2 other people’s arms had turned green in the area of lotion application.Day 10: After 10 applications, 2 more people’s arms had turned green in the area of lotion application.Day 15: After 15 applications, only one person’s arm still looked normal in the area of lotion application.Day 18: After 18 applications, the arms of all participants had turned green in the area of lotion application.

Skin Response to Daily Lotion Application

X axis: can be Days, # of Applications, or # of ObservationsY axis: # or % of Study Participants with Green Arm

Questions based on the results of the lotion study.

1. What day would be considered the NOEL? (no observed adverse effect level) Day 4

2. When is the ED50? (effective dose that affects 50% of the people) Day 10

3. When is maximal response obtained? Day 18



4. The graph you have completed (on page 1) is an “exposure-response” curve. What information do you need in order to prepare a toxicologist’s true “dose-response” curve?

A toxicologist’s “dose” requires knowing the weight (mg) of the lotion applied and the weight (kg) of each person to which it was applied.

Answer questions #5-12 based on the graph below.“Hypothetical toxicity results for two new cosmetic ingredients tested in mice”

5. What is the lethal dose for 50% of the mice (LD50) for ingredient A? 7 mg/kg For ingredient B? 7 mg/kg

6. What is the NOEL for ingredient A? 5 mg/kg For ingredient B? 2 mg/kg

7. Which compound is more toxic below the LD50? B Which compound is more toxic above the LD50? A

8. Does higher LD50 imply lower or higher toxicity? Lower toxicity

9. Once past the NOEL, how would you describe the response to increasing concentration (represented by the slope of the curve)? (circle your answer)

For ingredient A: slow medium fastFor ingredient B: slow medium fast

10. If this was a graph of ingredient irritation and both ingredients had the same NOEL, which “slope” would you prefer to see in a cosmetic and why.

B = there is a broader range of doses before 100% show irritation. Since people use varied amounts of cosmetics, you want less chance of irritation if they use more than the recommended amount.

11. If all of the mice received the same amount of ingredient but the mice were of very different ages and weights, where on the x axis would you find data for the light-weight mice?

To the right on the X-axis. Since all mice receive the same mg of ingredient, there is a higher ratio (mg/kg) in low weight mice.

12. Would you expect different responses (and therefore a different looking graph) from an ingredient that penetrated the skin to the dermis vs. one that remained in the epidermis? Explain your answer.

Yes, one would expect a difference. An ingredient that penetrates to the dermis could enter the bloodstream and be transported to many tissues. Any adverse effects in tissues would be greater in smaller mice (due higher dose [mg/kg]). If the ingredient stayed in the epidermis, there would be less effect related to weight. One might even see a straight line response – no difference in effect on mice of different weights.




File: Skin biology.doc NWABRJan. 2008

SUMMARY: Skin BiologyModified from http://www.cdc.gov/excite/skincancer/mod01.htm#skin

SKIN FUNCTIONS:Barrier to infection and injuryRegulates temperature, pH and water lossProtects against UV radiation from the sunMetabolism: produces Vitamin D, breaks down chemicalsSensory functions (pressure, pain, temperature)

Three major skin layers:

Epidermis - keratinocytesDermis - connective tissue with

blood vessels, glands, folliclesSubcutaneous layer – connective

tissue, fat

Approx. chemical composition of the skin:

Water 70.0% Protein 25.5% Lipids 2.0% Trace Minerals 0.5% Other 2.0%

Surface: 10-25% H2O, pH 4.2 - 5.6 Dermis: 70% H2O, pH 7.1 - 7.3

Picture from http://www.merck.com/mmhe/print/sec18/ch201/ch201b.html


Getting Under the Skin

File: Skin absorption.doc NWABRJan. 2008

Routes of skin absorption:

1. Around the cells – through extracellular lipids (MAJOR ROUTE)2. Through the cells3. Through hair follicles, sweat glands, and oil ducts

Epidermal Layers – composed of “keratinocytes”

15-20 cells thick.Non-living cells.(no nuclei, nometabolic func.;“corneocytes”)

Last layerof viable cells;have metabolizing enzymes.

Dividing (mitotic) cells are found in the basal layer and move upward anddifferentiate with age

Melanocytes: lie next to the basal layer; produce pigment and transfer it to the keratinocytes.

Langerhans cells: immune recognition cells; lie in epidermal layers

Source: www.who.int/ipcs/methods/draftehcdermalabsorption.pdf


File: Skin absorption.doc NWABRJan. 2008

Skin variations that can affect absorption:

1. Age (probably not gender or race)

2. Anatomical site – thickness varies

3. Temperature

4. Hydration of stratum corneum

5. Damage to stratum corneum

6. Metabolism in skin

Extracellular lipids are important!

o Form a barrier to water and polar compounds

o Become a route of entry for oils and non-polar compounds

o The lipid composition varies by anatomical site

Only compounds that make it to the dermis, can be absorbed into the blood stream.


File: cosmetic ingred devel.doc NWABRJan. 2008

Cosmetic ingredient development:

1. Literature search – previous safety tests - similar ingredients

2. Computer models of similar ingredients

3. Testing “in vitro” = test tubes, beakers

4. Testing on cells or tissues in culture

Research with animals to determine5. safe levels.

6. Human safety and quality testing


File: cosmetic devel activity.doc NWABR

Literature search - previous safety tests

- similar ingredients

Computer models of similar ingredients

Testing “in vitro” = test tubes, beakers

Testing on cells or tissues in culture

Research with animals to determine safe levels.

Human safety and quality tests

(TG) 3.15b

File: Drug devel process.doc NWABRJan. 2008

Drug development process:

1. Literature search – previous safety tests - similar ingredients

2. Computer models of similar ingredients

3. Testing “in vitro” = test tubes, beakers

4. Testing on cells or tissues in culture

5. Research with animals to determine safe levels

Manufacturing plan, label, clinical trial planIND submitted to FDA for approval (30 days)

6. Human safety and efficacy testing = Clinical TrialsPhase I, II, III

NDA submitted to FDA for approval (~1.5 yrs)

Phase IV – post-marketing studies

Northwest Association for (TG)3.16Biomedical Research Overhead

File: MSDS info - OH.doc NWABRJan. 2008

Material Safety Data Sheet (MSDS)

1. Purpose: an MSDS provides safety information to anyone who works with or comes in contact with the chemical

2. Availability: from chemical manufacturers web: http://hazard.com/msds

3. Information in an MSDS:

Section Title1 Chemical Product and Company Identification

NOTE: synonyms (one reason it is so difficult to identify

ingredients on product labels.)

2 Composition, Information on Ingredients

3 Hazards Identification

4 First Aid Measures

5 Fire Fighting Measures

6 Accidental Release Measures

7 Handling and Storage

8 Exposure Controls, Personal Protection

9 Physical and Chemical Properties

10 Stability and Reactivity

11 Toxicological Information

NOTE: results from animal studies12 Ecological Information

13 Disposal Considerations

14 Transport Information

15 Regulatory Information

16 Additional Information

Northwest Association for (TG)3.17Biomedical Research Overhead

File: MSDS info.docNWABR

Jan. 2008

Northwest Association for (TG)3.18Biomedical Research


Jan. 2008

Material Safety Data Sheet (MSDS)

1. Purpose: An MSDS provides safety information to anyone who works with or comes in contact with chemicals:

Chemists and other scientists in laboratoriesChemical manufacturersTransporters of chemicals

Any occupation that could come in contact with chemicalsjanitors, repairmen, firemen

They are especially important for the manufacturers of the chemicalbecause large quantities are handled and transported.The amount of information is overwhelming.

2. Availability: chemical manufacturers http://hazard.com/msds

Any company that sells a chemical, must supply an MSDS to the consumer.Company MSDSs are available to the public from companies or the web.

3. Information in an MSDS:

MSDSs from different companies are similar in format but the information may be different. Some have more information than others.

Section Title1 Chemical Product and Company Identification – section of interest

NOTE: synonyms (one reason it is so difficult to identify ingredients in products.)

2 Composition, Information on Ingredients 3 Hazards Identification4 First Aid Measures5 Fire Fighting Measures6 Accidental Release Measures7 Handling and Storage8 Exposure Controls, Personal Protection9 Physical and Chemical Properties10 Stability and Reactivity11 Toxicological Information – section of interest

NOTE: results from animal studies12 Ecological Information13 Disposal Considerations14 Transport Information



Jan. 2008

15 Regulatory Information16 Additional Information


File: MSDS tox info.docNWABR

Jan. 2008

Information about Section 11 in an MSDS

Within Section 11, you will see the results of animal testing.1. Species and routes of administration

Results include animal species, route of administration and results(Format and results may be slightly different between different MSDSs.)

The safety of chemicals are usually tested by exposing animals to the chemical in various ways. (orally, topically on the skin, injection)

The routes of administration reflect the many ways that humans might be exposed to a chemical – through the skin, the mouth, the lungs, or an injection. Remember that in chemical manufacturing plants, workers could get exposed to large quantities so understanding the limits of safe concentrations is important.

2. Toxicity results are usually presented as LD50 or LC50LD50 – the lethal dose at which 50% of animals dieLC50 – the lethal concentration at which 50% of animals die

The original LD50 tests have been replaced with tests that use fewer animals.“Limit tests” are done instead. Toxicologists start at low doses and test a few animals at

a time. If animals survive, then they go to increasingly higher doses on small numbers of animals at a time.

A low LD50 means the compound is highly toxic.LD50 doesn’t tell you about non-lethal toxic effects, such as redness, irritation, swelling, etc.

Draize eye test: a substance is placed in the eye of an albino rabbit to measure the reaction induced after 24 hours.

The original Draize test protocol has been modified. Compounds are not tested at concentrations that are expected to be toxic. Analgesics may be used. This in vivo test is used after compounds have been screened in other non-whole animal assays. (see R3.6)

3. Extrapolation to humansAnimals and humans share many infectious diseases, as well as chronic, non-infectious

diseases because of similar physiology. Different species are better models for different diseases. Tests are done in animal species that are most similar to humans for that particular test.

Example: Pigs are used for heart studies and skin penetration studies Rabbits are used for glaucoma studies (inherited in NZW rabbits) Transgenic or gene-knockout animals may be used because they closely mimic human diseases



Jan. 2008

Test results from animals can be extrapolated to humans. There are conversion factors to adjust for the fact that metabolism rates or skin penetration rates differ between animals and humans.

Converting Animal Results to Humans

Results from toxicity tests in animals can be extrapolated to humans to give estimated toxicities. For greater accuracy, species-specific conversion factors are available to account for the fact that the basic biology (e.g., metabolism rates, skin penetration, etc.) differs between animals and humans.

EXAMPLE: LD50 value from a mouse

Substance: Triethanolamine (TEA)

Results: Oral delivery to a mouseLD50 = 5846 mg/kg

MATHEMATICAL CONVERSION of mouse LD50 to a human LD50

based on weight, without use of species-specific conversion factors:

1 kilogram (kg) = 1000 grams (g) = 2.2 pounds1 milligram (mg) = 0.001 gram

Average mouse = 20-25 grams = 0.02-0.025 kg Average rat = 0.25-0.40 kgAverage human = 154 pounds = 70 kg Average rabbit = 4-6 kg

For a 20 g mouse: oral LD50 = 5846 mg/kg x 0.02 kg/mouse = 116.92 mg / mouse

For a 70 kg human: 5846 mg/kg x 70 kg/human = 409,220 mg / human

1000 mg / gram, therefore 409,220 mg = 409.22 grams / human

How many pounds is 409.22 grams?

1 pound (avdp) = 453.59 grams409.22 grams / 453.59 grams/pound = 0.9 pounds

Therefore, by straight conversion based on species’ weight:

Oral LD50 for triethanolamine (TEA)

Human (70 kg) = 409,220 mg = 0.9 poundsNorthwest Association for (TG)3.19Biomedical Research


Jan. 2008

Mouse (20 g) = 117 mg = 0.00026 pounds

Therefore, 0.9 pounds of TEA would be lethal to 50% of the humans who ate it.That is a lot of TEA!


File: Animal claims.doc NWABRAug. 2008

Lotion Ingredient

Ingredient MSDSon labels

Claim on label: Animals used;

“Not tested in animals” ≠ multiple delivery modes

Do consumers really understand what that claim means?

Northwest Association for Teacher Guide (TG) 3.21

Teacher Guide (TG)3.20

Biomedical Research. Overhead


“Not tested in animals.”

How can companies make this claim?

1. Individual ingredients were tested but not the final product.

2. Someone else did the testing.

3. Animal tests were done more than 5 years ago.

4. No new, unique ingredients therefore new animal safety testing is not needed.








File: Animal studies bkgd - OH.doc NWABRJan. 2008

Northwest Association for Teacher Guide (TG) 3.22 Biomedical Research. Overhead

File: Animal studies bkgd - OH.doc NWABRJan. 2008

Animal Studies - Background

1. New ingredients for cosmetics and drugs are required to be safe.

At this time, some aspects of safety can only be obtained through responsible studies with animals.

2. When testing is required on new compounds, animal studies are done after gathering data from other sources

3. When animal studies are needed, the guiding principles are the 3 R’s

ReplacementReduction Refinement

4. Federal regulations: govern care, research, housing, transport

a. Animal Welfare Act, 1966; amended 1970, 1985, 1990

b. Public Health Service Policy based on Health Research Extension Act, 1985

5. Animal study proposals must be approved by site committees before they can be started

6. To replace an animal test with a new test requires “validation”


File: Animal studies discussion - OH.doc NWABRJan. 2008

Animal Studies - Background

1. New ingredients for cosmetics and drugs must be safe. Animal studies are needed.The cosmetic safety studies do not have to be approved by FDA before marketingNo alternative tests exist for some animal safety tests and for understanding ingredient distribution, metabolism, and excretion.

2. When testing is required on new compounds, animal studies are done after gathering data from other sources:

Literature searches for information about similar compoundsMathematical and computer based modeling from available data basesIn vitro (in glass) testing (for example, effect on a specific target enzyme)Cell and tissue culture (non-human or human cells or tissues; normal or cancerous cells)

3. When animal studies are needed, the guiding principles are the 3 R’sReplacement = using non-animal models when appropriateReduction = using the minimum number of animals necessaryRefinement = enhancing animal welfare and ensuring the best conditions possible

4. Federal regulations:a. Animal Welfare Act, 1966; amended 1970, 1985, 1990. Regulates transport, sale, and handling of all warm-blooded animals (not, mice or rats) Requires registration with, and inspections by, USDA Requires review and approval of all proposed animal studies

c. Public Health Service Policy based on Health Research Extension Act, 1985Applies to publicly funded researchCovers all vertebrate animals, including fish, reptiles, rats, mice

5. Animal study proposals must be approved before they can be startedInstitutional Animal Care and Use Committee (IACUC) = composed of

veterinarian, researcher, non-scientist professional, community memberReviews protocols and inspects facilities

Protocol review includes: Literature search to show that a similar study has not been doneJustification for using animals and the species chosenUse of appropriate number of animals - to get statistically significant resultUse of appropriate procedures to reduce pain Personnel appropriately trained

6. To replace an animal test with a new test requires “validation”Multiple US agencies must evaluate the new procedureA new test must be repeatable by multiple labs and results must be

reproducibleThe results must correlate with previously acquired data

Northwest Association for Teacher Guide (TG) 3.23 Biomedical Research.

File: Animal studies discussion - OH.doc NWABRJan. 2008


File: IACUC.doc NWABRJan. 2008

Animal study protocols must be approved in advance by the

Institutional Animal Care and Use Committee (IACUC)

WHO are they: The committee must include at least 3-5 members, including:

VeterinarianResearcher – experienced in research with animalsNon-scientist professional – such as lawyer, clergyCommunity Member – not affiliated with the institution

FUNCTION: Review protocols and personnel qualifications Post-approval monitoring Facility review

The animal study protocol must include:

1. Literature search - to show that a similar study has not been done

2. Justification for using animals and the species chosen

3. Use of appropriate number of animals – to get statistically significant result

4. Use of appropriate procedures to reduce pain

5. Documentation that shows personnel have been appropriately trained


File: 3Rs - OH.doc NWABRJan. 2008

Research with Animals: Practices and Procedures

People who work with animals: Are passionate about high standards of care for animals Take animal studies very seriously Use animals in research only after institutional approval

The guiding principles for using fewer animals are summed up in the

3 R’s

Replacement – whenever possible, replace animal testing with methods that don’t use whole animals.

Reduction – use methods that reduce the number of animals necessary to get the same information

Refinement – use procedures to eliminate or minimize discomfort and improve animal well-being


File: 3Rs - disc.doc NWABRJan. 2008

Research with Animals: Practices and Procedures

The goal of all people who work with animals is to use as few animals as possible and to replace animal testing with non-animal testing when possible. Maintaining high standards of animal care ensures that study results will be more accurate and reproducible.

Currently, in vitro (in test tube or non-animal) tests are not able to replace all animal safety tests. Only a live animal can provide the dynamic environment of multiple cell types interacting and responding to the environment.

The guiding principles for using fewer animals are summed up in the 3 R’s.

Replacement – whenever possible, replace animal testing with methods that don’t use whole animals.

EXAMPLES: a. In the past, rabbits were used for pregnancy tests. Technicians injected a urine extract from a woman into a rabbit and later tested the rabbit to see if the injection had caused the rabbit to ovulate, indicating a positive test for pregnancy. Now, test strips can test for hormones in human urine.b. Tests for bacteria that could cause fevers were done by injecting the test compound into rabbits and taking their temperature 24 hours later. Now, the “blood” of horseshoe crabs is used to test for fever-inducing bacteria (LAL assay) c. Human blood samples are incubated with the test compound and then tested for the production of proteins called cytokines, which signal the brain to initiate a fever.

Reduction – use methods that reduce the number of animals necessary to get the same information

EXAMPLES: a. By referring to data bases or using computer modeling, predictions can be made about the safety of some compounds. Very acidic compounds are known to be irritating to skin so there is no need to test those compounds at concentrations that are known to be problematic.b. Biophotonics uses light to detect and measure changes in biological tissues (such as tumor growth) without sacrificing animals.c. Species lower on the evolutionary ladder are used in assays when possible = yeast, fish, fruit flies, or worms (nematodes).

Refinement – use procedures to eliminate or minimize discomfort and improve animal well-beingEXAMPLES: a. New and more effective anesthetics and analgesics are being developed. b. Animals are housed in groups since that is less stressful. c. Animals are provided “toys” to alleviate boredom and make their environment more natural.


File: Human study example.doc NWABRJan. 2008

Description of a human lotion study:

I gave the lotion to my friends to use.

They thought it was fine so I believe it is

OK to put on the market.



A well-designed study (scientific investigation)

Must be informative - with a clear purpose, - with 1 variable that is changed

(manipulated or independent variable)

- with other variables kept the same (constants or controlled variables)

- with variable(s) that are measured(responding or dependent variable(s))

Must be reliable - objective data collection and measurable results

Must be reproducible

Scientific Method: Information found in a lab report

Purpose

Hypothesis Testable and measurable

Methods MaterialsProcedure ** enough details for

reproducibility

Results ObservationsMeasurable dataData analysis

Conclusion



Details of a human lotion study:

WHAT

WHO WHEN WHERE

I gave the lotion to my friends to use.

They thought it was fine so I believe it is

OK to put on the market.

HOW evaluate

PURPOSE


File: Lotion study basics - OH.doc NWABRJan. 2008

Lotion Study Basics

An informative, reliable, and reproducible lotion study must be well designed.

Use these basic categories to help you design a human safety or quality study for your lotion. Identify the controlled, the manipulated (independent) and responding variables.

PURPOSE:

WHO:

WHAT:

WHEN:

WHERE:

HOW will the results be evaluated?


File: Lotion study discuss.doc NWABRJan. 2008

Designing a Lotion Study

PURPOSE: What aspect of safety is being studied?What qualities of the lotion are desirable or undesirable?

Study Design ConsiderationsWHO: Who can be in your study? Age, gender, race, health

Is a consent form needed?How many people will participate?

WHAT: What will be tested?Will subjects compare your product against a commercially available product?Will subjects know which product they are testing? Will the investigator know which product subjects are testing?

If the study is testing 2 products, does each subject get to test both products? Sequentially? Simultaneously (for example: one product per arm)?

What method of application and by whom – investigator or subject?What amount should be applied and where?

WHEN: How often will the test article be applied?What time of day will the test article be applied?How long does the test article have to be left on (before being washed off)?How long will the study last?

HOW will the results be evaluated: Who will record the results and how?

Verbal interview Daily diary Written evaluation at end using rating scales, multiple choice, narrativePictures - before and after

When will the results be recorded – daily, periodically, or end of study?Who will tabulate and summarize the results?

Do you have to repeat the study for confidence (statistical significance) in the results? Did you include enough people for the results to be representative ofyour intended consumers?

Identify the controlled variables and manipulated (independent) and responding variables in your study.


File: Lotion dose response wksht.doc NWABRJuly 2008

Evaluation Rubric: Winning Combination: Superb Lotion Study and Eye-Catching Label

Name: Date: Period:

Concept Exceeds Expectations Meets Expectations Developing; Some Expectations Met

Below Expectations Score

Drug vs. Cosmetic

Product is correctly identified as a cosmetic or drug. The justification for the designation chosen draws upon exceptional understanding of product regulation.

Product is correctly identified as a cosmetic or drug, with an accurate justification of designation chosen.

Product is correctly identified as a cosmetic or drug, but the justification of designation chosen is unclear or incomplete.

Product is incorrectly identified as a cosmetic or drug, and/or the justification of the designation chosen is rudimentary or absent.

Animal Test Describes the relation of the product to animal testing with insight, demonstrates exceptional understanding of current regulations and the need for testing.

Describes the relation of the product to animal testing, demonstrates understanding of current regulations and the need for testing.

Describes the relation of the product to animal testing, but relation may be unclear or incomplete. Demonstrates some understanding of current regulations and the need for testing.

Is unable to describe the relation of the product to animal testing. Does not reference current regulations or the need for testing.

Claims Provides 2 or more claims. Demonstrates in-depth understanding of claims that can be made on labels. Label claims are accurate.

Provides 2 claims. Demonstrates understanding of claims that can be made on labels. Label claims are accurate.

Demonstrates some understanding of claims that can be made on labels. Label claims are mostly accurate.

Demonstrates little understanding of claims that can be made on labels. Label claims are inaccurate or irrelevant

Northwest Association for Student (S)3.1Biomedical Research.


Human Lotion Test

Describes 2 or more features of each of the study design elements (Who, What, When, Reliable Results) Study design is exceptionally thoughtful, clear, and logical.

Describes 2 features of each of the study design elements. Study design is clear and logical.

Describes fewer than 2 features for some of the study design elements below. Study design may be incomplete or unclear.

Few of the study design elements are described. Study design is rudimentary or absent.

Label Information

Label information is clearly and accurately presented, with product name, ingredients listed in proper order (from those present in largest volume to least), manufacturing address, and reasonable estimate of quantity included.

Label information is accurately presented, with product name, ingredients listed in proper order, manufacturing address, and a reasonable estimate of quantity included.

Label information is partially presented. Label ingredients are included but may not be correctly ordered. The manufacturing address may be incomplete or missing. Product name may be missing. Estimated quantity may be misjudged.

Label information is missing most required elements: Label ingredients, manufacturing address, and product name, estimated quantity.

Label Design

The label is exceptionally well-designed, with all elements attractively presented.

The label has all the required elements clearly presented.

The required elements are not presented in a clear way.

The label design detracts from the elements, or the elements are not presented in the design.



Dose-Response Worksheet: How much is too much?

Name __________________________ Period _________ Date __________

Hypothetical: Human Safety Test of a New Lotion

A local cosmetic manufacturer has developed a new moisturizing lotion. The lotion has passed all of the animal-based safety tests and is now ready for testing on humans. Healthy males and females have been recruited for the study. They have been fully informed about the protocol and the risks and have voluntarily signed informed consent forms.

Ten people are participating in the study. Each person will be given a pre-measured, single use packet of lotion to rub on their right, inside forearm. Each day for 20 days, they will come to the clinic and be given a new packet of lotion to apply to their right, inside forearm. A clinical research associate (CRA) will observe the arm and record skin appearance before application of the next packet of lotion.

Following is a summary of the results of the study based on the CRA’s observations. Please graph the results below, labeling the axes appropriately.

Day 0: All 10 arm surfaces looked normal and healthyDays 1-4: After 4 applications, all 10 arm surfaces look normal and healthyDay 5: After 5 applications, 1 person’s arm had turned green in the area of lotion applicationDay 7: After 7 applications, 2 other people’s arms had turned green in the area of lotion application.Day 10: After 10 applications, 2 more people’s arms had turned green in the area of lotion application.Day 15: After 15 applications, only one person’s arm still looked normal in the area of lotion application.Day 18: After 18 applications, the arms of all participants had turned green in the area of lotion application.

Skin Response to Daily Lotion Application

____________________________________________

Questions based on the results of the lotion study.

1. What day would be considered the NOEL ? (no observed adverse effect level)

2. When is the ED50? (effective dose that affects 50% of the people)

3. When is maximal response obtained?



4. The graph you have completed (on page 1) is an “exposure-response” curve. What information do you need in order to prepare a toxicologist’s true “dose-response” curve?

Answer questions #5-12 based on the graph below.“Hypothetical toxicity results for two new cosmetic ingredients tested in mice.”

5. What is the LD50 for ingredient A? ____________ For ingredient B? _________

6. What is the NOEL for ingredient A? ___________ For ingredient B? _________

7. Which compound is more toxic below the LD50? Which compound is more toxic above the LD50?

8. Does higher LD50 imply lower or higher toxicity?

9. Once past the NOEL, how would you describe the response to increasing concentration (represented by the slope of the curve)? (circle your answer)

For ingredient A: slow medium fastFor ingredient B: slow medium fast

10. If this was a graph of ingredient irritation and both ingredients had the same NOEL, which “slope” would you prefer to see in a cosmetic and why.

11. If all of the mice received the same amount of ingredient but the mice were of very different ages and weights, where on the x axis would you find data for the light-weight mice?

12. Would you expect different responses (and therefore a different looking graph) from an ingredient that penetrated the skin to the dermis vs. one that remained in the epidermis? Explain your answer.


File: Func, tox chart.doc NWABRFeb. 2005

Northwest Association for Biomedical Research. Student (S)3.2 Handout




This page was intentionally left blank.


File: Winning combination.doc NWABRJan. 2008

Winning Combination: Superb Lotion Study and Eye-Catching Label

Name: __________________________ Date: _______________ Period: _______

1. Evaluate your lotion:Is the lotion a cosmetic or a drug? Explain your answer.

Is animal testing required on your lotion? Explain your answer.

2. List at least two claims for your label. Explain why you can make these claims

Northwest Association for Students (S)3.3 Biomedical Research Homework

File: Winning combination.doc NWABRJan. 2008

Winning Combination: Superb Lotion Study and Eye-Catching Label

3. Design a human study for the safety or quality of the skin care lotion that you made in class. The study design should address the following points: Except for “Purpose”, include at least 2 features of your study design under each point.

Purpose:

Who:

What:

When:

Where:

How will results be evaluated?

4. On a separate page, design a label for your lotion that includes claims and information required for all cosmetic labels: product name, ingredients listed in decreasing order of quantity, manufacturer’s address, product quantity (estimated)

Northwest Association for Students (S)3.3 Biomedical Research Homework

File: L3 websites.doc NWABRJuly 2008

Northwest Association for Resources (R)3.1Biomedical Research


Lesson 3 Website ResourcesSkin Biology

1. http://www.cdc.gov/excite/skincancer/mod01.htm#skinBasic explanations of skin biology

2. http://www.who.int/ipcs/methods/draftehcdermalabsorption.pdf“Dermal Absorption”, International Programme on Chemical Safety Environmental Health Criteria, Draft Feb. 2005Excellent descriptions of skin function and dermal absorption testing

3. http://www.nature.com/nature/supplements/insights/skin/index.htmlSkin Biology Supplement: melanocytes, skin development, pigmentation, sensory transduction, psoriasis, tissue engineering.

4. http://www.nlm.nih.gov/medlineplus/medlineplus.html“Medline Plus” from NIH and US National Library of MedicineSections on health topics, medical encyclopedia, other resources

Material Safety Data Sheets

1. http://hazard.com/msds Find MSDS’s by company or compound name. Toxicology reports searchable by compound name.

2. http://www.msdssearch.comBackground information about MSDS’s. Directory of MSDS’s by ingredient and manufacturer.

3. http://www.ilpi.com/msds/References to MSDS information sites. Contains a glossary of terms found in MSDS’s.

4. http://www.householdproducts.nlm.nih.govSelected personal care/use products and ingredients have summaries taken from MSDSsheets provided by manufacturers. Specific animal toxicology testing information is notincluded.

Toxicology Information 1. http://toxnet.nlm.nih.gov

TOXNET contains databases on toxicology, hazardous chemicals, environmental health, and toxic releases

2. http://toxipedia.org/conf/display/toxipedia/Welcome+to+Toxipedia Toxipedia – Great site devoted to providing a better understanding of many aspects of toxicology; includes a

toxic substances dictionary, teaching resources, lists of agencies and organizations involved in health and environmental protection.

3. http://www.atsdr.cdc.gov/COM/exposure.pdfThe Agency for Toxic Substances and Disease Registry is a federal public health agency of the US Dept. of Health and Human Services. This is a two page explanation of “exposure”. Although

primarily focused on environmental health, the main web site has a nice glossary of terms for the broad area of toxicology.


http://www.ilpi.com/msds/

http://hazard.com/msds

http://www.nature.com/nature/supplements/insights/skin/index.html

http://www.cdc.gov/excite/skincancer/mod01.htm#skin


4. http://whs.wsd.wednet.edu/Faculty/Young/YNHC/ynhc.htmThe Youth Network for Healthy Communities program was developed by UW Center for Ecogenetics and Environmental Health. See Resources -> Orientation Power Point and Resource List

Product Safety Testing1. http://www.kids4research.org/productsafety.html

Product safety testing information and glossary; animals in research information

2. http://www.nabr.org /issues.htm “The use of animals in product safety testing”, 1997See copy of article under Tab 8.

3. http://www.cosmeticsdesign-europe.com/news/ng.asp?n=78158-ecvam-eu-animal-sccp-colipaNews article about animal testing in Europe and REACH effects, 7-16-07

4. http://www.fda.gov/oc/history/historyoffda/default.htmHistory of the Food and Drug Administration

5. http://www.cosmeticsdesign-europe.com/news/ng.asp?n=78158-ecvam-eu-animal-sccp-colipa News article about animal testing in Europe and REACH effects, 7-16-07

6. http://www.fda.gov/oc/history/historyoffda/default.htmHistory of the Food and Drug Administration

Alternatives to Animal Testing1. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM); responsible for testing the accuracy and reliability of test protocols. National Toxicology Program, Dept. of Health and Human Services

Alternative animal tests for dermal corrosivity and irritation http://iccvam.niehs.nih.gov/methods/dermal/dermal.htm

Alternatives to animal testing for ocular toxicityhttp://iccvam.niehs.nih.gov/methods/ocutox/ocutox.htm

In vitro pyrogen test methodshttp://iccvam.niehs.nih.gov/methods/pyrogen/pyrogen.htm

Alternatives to animal testing for acute oral toxicityhttp://iccvam.niehs.nih.gov/methods/acutetox/acutetox.htm

2. http://altweb.jhsph.edu/ Johns Hopkins School of Public Health

3. http://caat.jhsph.edu/ Johns Hopkins Center for Alternatives to Animal Testing

4. http://ecvam.jrc.cec.eu.int/index.htmEuropean Centre for the Validation of Alternative Methods (ECVAM)


http://caat.jhsph.edu/

http://altweb.jhsph.edu/

http://iccvam.niehs.nih.gov/methods/acutetox/acutetox.htm

http://iccvam.niehs.nih.gov/methods/pyrogen/pyrogen.htm

http://iccvam.niehs.nih.gov/methods/ocutox/ocutox.htm

http://iccvam.niehs.nih.gov/methods/dermal/dermal.htm

http://www.cosmeticsdesign-europe.com/news/ng.asp?n=78158-ecvam-eu-animal-sccp-colipa

http://www.cosmeticsdesign-europe.com/news/ng.asp?n=78158-ecvam-eu-animal-sccp-colipa

http://www.nabr.org/

File: Dose-response bkgd.doc NWABRJuly 2008

http://pmep.cce.cornell.edu/profiles/extoxnet/TIB/dose-response.html Viewed 8-10-07PMEP Home Page

Pesticide Active Ingredient Information

EXTOXNET: The Extension Toxicology Network

Toxicology Information Briefs

Dose-Response Relationships In Toxicology

E X T O X N E T Extension Toxicology Network

A Pesticide Information Project of Cooperative Extension Offices of Cornell University, Michigan State University, Oregon State University, and University of California at Davis. Major support and funding was provided by the

USDA/Extension Service/National Agricultural Pesticide Impact Assessment Program.

Toxicology

Information

Brief

Dose-Response Relationships In Toxicology

Publication Date: 9/93 "The right dose differentiates a poison and a remedy." -Paracelsus

INTRODUCTIONThe science of toxicology is based on the principle that there is a relationship between a toxic reaction (the response) and the amount of poison received (the dose). An important assumption in this relationship is that there is almost always a dose below which no response occurs or can be measured. A second assumption is that once a maximum response is reached any further increases in the dose will not result in any increased effect. One particular instance in which this dose-response relationship does not hold true, is in regard to true allergic reactions. Allergic reactions are special kinds of changes in the immune system; they are not really toxic responses. The difference between allergies and toxic reactions is that a toxic effect is directly the result of the toxic chemical acting on cells. Allergic responses are the result of a chemical stimulating the body to release natural chemicals which are in turn directly responsible for the effects seen. Thus, in an allergic reaction, the chemical acts merely as a trigger, not as the bullet.

For all other types of toxicity, knowing the dose-response relationship is a necessary part of understanding the cause and effect relationship between chemical exposure and illness. As Paracelsus once wrote, "The right dose differentiates a poison from a remedy." Keep in mind that the toxicity of a chemical is an inherent quality of the chemical and cannot be changed without changing the chemical to another form. The toxic effects on an organism are related to the amount of exposure.

MEASURES OF EXPOSURE

Exposure to poisons can be intentional or unintentional. The effects of exposure to poisons vary with the amount of exposure, which is another way of saying "the dose." Usually when we think of dose, we think in terms of taking one vitamin capsule a day or two aspirin every four hours, or something like that. Contamination of food or water with chemicals can also provide doses of chemicals each time we eat or drink. Some commonly used measures for expressing levels of


http://pmep.cce.cornell.edu/profiles/extoxnet/TIB/index.html

http://pmep.cce.cornell.edu/profiles/extoxnet/TIB/index.html

http://pmep.cce.cornell.edu/profiles/extoxnet/index.html

http://pmep.cce.cornell.edu/profiles/extoxnet/index.html

http://pmep.cce.cornell.edu/profiles/index.html

http://pmep.cce.cornell.edu/profiles/index.html

http://pmep.cce.cornell.edu/

http://pmep.cce.cornell.edu/

http://pmep.cce.cornell.edu/profiles/extoxnet/TIB/dose-response.html


contaminants are listed in table 1. These measures tell us how much of the chemical is in food, water or air. The amount we eat, drink, or breathe determines the actual dose we receive.

Concentrations of chemicals in the environment are most commonly expressed as ppm and ppb. Government tolerance limits for various poisons usually use these abbreviations. Remember that these are extremely small quantities. For example, if you put one teaspoon of salt in two gallons of water the resulting salt concentration would be approximately 1,000 ppm and it would not even taste salty!

Table 1. Measurements for Expressing Levels of Contaminants in Food and Water.Dose Abbrev. Metric equivalent Abbrev. Approx. amt. in water

parts per million ppm milligrams per kilogram mg/kg 1 teaspoon per 1,000 gallonsparts per billion ppb micrograms per kilogram ug/kg 1 teaspoon per 1,000,000 gallons

DOSE-EFFECT RELATIONSHIPS

The dose of a poison is going to determine the degree of effect it produces. The following example illustrates this principle. Suppose ten goldfish are in a ten-gallon tank and we add one ounce of 100-proof whiskey to the water every five minutes until all the fish get drunk and swim upside down. Probably none would swim upside down after the first two or three shots. After four or five, a very sensitive fish might. After six or eight shots another one or two might. With a dose of ten shots, five of the ten fish might be swimming upside down. After fifteen shots, there might be only one fish swimming properly and it too would turn over after seventeen or eighteen shots. The effect measured in this example is swimming upside down. Individual sensitivity to alcohol varies, as does individual sensitivity to other poisons. There is a dose level at which none of the fish swim upside down (no observed effect). There is also a dose level at which all of the fish swim upside down. The dose level at which 50 percent of the fish have turned over is known as the ED50, which means effective dose for 50 percent of the fish tested. The ED50 of any poison varies depending on the effect measured. In general, the less severe the effect measured, the lower the ED50 for that particular effect. Obviously poisons are not tested in humans in such a fashion. Instead, animals are used to predict the toxicity that may occur in humans.

One of the more commonly used measures of toxicity is the LD50. The LD50 (the lethal dose for 50 percent of the animals tested) of a poison is usually expressed in milligrams of chemical per kilogram of body weight (mg/kg). A chemical with a small LD50 (like 5 mg/kg) is very highly toxic. A chemical with a large LD50 (1,000 to 5,000 mg/kg) is practically non-toxic. The LD50 says nothing about non-lethal toxic effects though. A chemical may have a large LD50, but may produce illness at very small exposure levels. It is incorrect to say that chemicals with small LD50s are more dangerous than chemicals with large LD50s, they are simply more toxic. The danger, or risk of adverse effect of chemicals, is mostly determined by how they are used, not by the inherent toxicity of the chemical itself.

The LD50s of different poisons may be easily compared; however, it is always necessary to know which species was used for the tests and how the poison was administered (the route of exposure), Northwest Association for Resources (R)3.2Biomedical Research


since the LD50 of a poison may vary considerably based on the species of animal and the way exposure occurs. Some poisons may be extremely toxic if swallowed (oral exposure) and not very toxic at all if splashed on the skin (dermal exposure). If the oral LD50 of a poison were 10 mg/kg, 50 percent of the animals who swallowed 10 mg/kg would be expected to die and 50 percent to live. The LD50 is determined mathematically, and in actual tests using the LD50, it would be unusual to get an exact 50% response. One test might produce 30% mortality and another might produce 70% mortality. Averaged out over many tests, the numbers would approach 50%, if the original LD50 determination was valid.

The potency of a poison is a measure of its strength compared to other poisons. The more potent the poison, the less it takes to kill; the less potent the poison, the more it takes to kill. The potencies of poisons are often compared using signal words or categories as shown in the example in table 2.

The designation toxic dose (TD) is used to indicate the dose (exposure) that will produce signs of toxicity in a certain percentage of animals. The TD50 is the toxic dose for 50 percent of the animals tested. The larger the TD the more poison it takes to produce signs of toxicity. The toxic dose does not give any information about the lethal dose because toxic effects (for example, nausea and vomiting) may not be directly related to the way that the chemical causes death. The toxicity of a chemical is an inherent property of the chemical itself. It is also true that chemicals can cause different types of toxic effects, at different dose levels, depending on the animal species tested. For this reason, when using the toxic dose designation it is useful to precisely define the type of toxicity measured, the animal species tested, and the dose and route of administration.

Table 2. Toxicity Rating Scale and Labeling Requirements for Pesticides.

Category Signal word required on label

LD50 oral mg/kg(ppm)

LD50 dermal mg/kg(ppm)

Probable oral lethal dose

Ihighly toxic

DANGER-POISON(skull and crossbones) less than 50 less than 200 a few drops to a

teaspoonIImoderately toxic WARNING 51 to 500 200 to 2,000 over 1 teaspoon to 1

ounceIIIslightly toxic CAUTION over 500 over 2,000 over 1 ounce

IVpractically non-toxic

none required

Toxicity assessment is quite complex, many factors can affect the results of toxicity tests. Some of these factors include variables like temperature, food, light, and stressful environmental conditions. Other factors related to the animal itself include age, sex, health, and hormonal status.

The NOEL (no observable effect level) is the highest dose or exposure level of a poison that produces no noticeable toxic effect on animals. From our previous fish example, we know that there is a dose below which no effect is seen. In toxicology, residue tolerance levels of poisons that are permitted in food or in drinking water, for instance, are usually set from 100 to 1,000 times less than the NOEL to provide a wide margin of safety for humans.



The TLV (threshold limit value) for a chemical is the airborne concentration of the chemical (expressed in ppm) that produces no adverse effects in workers exposed for eight hours per day five days per week. The TLV is usually set to prevent minor toxic effects like skin or eye irritation.

Very often people compare poisons based on their LD50's and base decisions about the safety of a chemical based on this number. This is an over-simplified approach to comparing chemicals because the LD50 is simply one point on the dose-response curve that reflects the potential of the compound to cause death. What is more important in assessing chemical safety is the threshold dose, and the slope of the dose-response curve, which shows how fast the response increases as the dose increases. Figure 1 shows examples of dose-response curves for two different chemicals which have the same LD50. Which of these chemicals is more toxic? Answer this question for doses below the LD50 and it is chemical A which is more toxic, at the LD50 they are the same, and above the LD50, chemical B is more toxic. While the LD50 can provide some useful information, it is of limited value in risk assessment because the LD50 only reflects information about the lethal effects of the chemical. It is quite possible that a chemical will produce a very undesirable toxic effect (such as reproductive toxicity or birth defects) at doses which cause no deaths at all.

A true assessment of chemical toxicity involves comparisons of numerous dose-response curves covering many different types of toxic effects. The determination of which pesticides will be Restricted Use Pesticides involves this approach. Some Restricted Use Pesticides have very large LD50s (low acute oral toxicity), however, they may be very strong skin or eye irritants and thus require special handling.

The knowledge gained from dose-response studies in animals is used to set standards for human exposure and the amount of chemical residue that is allowed in the environment. As mentioned previously, numerous dose-response relationships must be determined, in many different species. Without this information, it is impossible to accurately predict the health risks associated with chemical exposure. With adequate information, we can make informed decisions about chemical exposure and work to minimize the risk to human health and the environment.

Revised 9/93


File: Skin layers.doc NWABRAug. 2007

Modified from http://www.cdc.gov/excite/skincancer/mod01.htm#skin

EPIDERMIS

The epidermis is made up of several different types of cells. Two of these cell types are important in studying skin cancer: keratinocytes and melanocytes. Langerhans’ cells are involved in protecting the body from foreign substances and they initiate the skin’s allergic reactions.

KeratinocytesThe main cells that make up the epidermis are called keratinocytes. These cells are arranged

into five layers, or "strata." basal layer (stratum basale), with dividing keratinocytes Deepest layer "prickle-cell" layer (stratum spinosum), granular layer (stratum granulosum), lucid layer (stratum lucidum), and cornified layer (stratum corneum). Top, surface

Basal LayerThe basal layer is the deepest layer. Cells in this layer can divide and reproduce themselves. They are the most immature, or least specialized, of the keratinocytes. These cells mature and move toward the surface of the skin. As they move upward, they become flattened and lose most of their water content.

Cornified LayerThe stratum corneum is the outermost (top) layer, composed mainly of dead keratinocytes, hardened proteins (keratins) and lipids, forming a protective crust. It has the flattest cells, arranged in a basket weave pattern. Keratin in these cells helps the cornified layer protect against moisture, light, and infection. The keratin works together with lipids (fats) and tight inter-cellular connections called "desmosomes." The cells of the stratum corneum are the most specialized of the keratinocytes. These dead cells are continually sloughed off the surface of the skin and replaced by cells that mature from the basal layer. The skin completely renews itself every 3-5 weeks.

MelanocytesMelanocytes are another kind of cell found in the epidermis. These cells are in the basal

layer. Melanocytes make the pigment called melanin. The melanin is then transferred to nearby keratinocytes. Some of this melanin then works its way up with the keratinocytes as they move to the top layer.

Skin color depends on how much melanin is made and how much is carried toward the surface. All people have about the same number of melanocytes, no matter what their skin color. Those with darker skin have more melanin in the upper layers of the skin. Sunlight raises the rate at which melanin is made and transferred. This is what causes tanning.

Melanin can protect the cells from ultraviolet (UV) radiation and its harmful effects. It does this by absorbing harmful UV rays. It also cleans up toxins that come from UV damage to skin


File: Skin layers.doc NWABRAug. 2007

cells. Within cells, melanin tends to form caps above the nuclei. The caps protect the cells' genetic material from UV damage.

Langerhans’ CellsThese epidermal cells are part of the skin’s immune system. They help detect foreign

substances and play a role in the development of skin allergies.

DERMISThe dermis (2-3 mm thick) lies under the epidermis and is much thicker than the epidermis

(< 1mm). It is made of loosely arranged connective tissue. The surface of the dermis is covered with rows of projections much like fingers. These are called dermal papillae. They help hold the epidermis and dermis together by forming ridges and grooves.

The lower part of the dermis is a network of interlacing fibers. These fibers give the skin strength and allow it to stretch. The dermis contains a lot of blood vessels, nerve endings, muscle fibers, sweat and oil (sebaceous) glands, and hair follicles. The roots of hairs are located deep in follicles. Hair shafts stick out from the follicles. Small muscles raise and lower the hairs. These muscles extend from the dermis to the side of the hair follicles. Sebaceous glands open into hair follicles and secrete oil called sebum. Sebum oils the skin and hair.

SUBCUTANEOUS LAYER

Subcutaneous tissue lies under the dermis. It is a thick layer of connective tissue and fat. The fat helps control body temperature and stores energy from food. The subcutaneous tissue also acts as a shock absorber. It protects the tissues below from injury.


File: Drug devel process.doc NWABRJan. 2008

Summary of the drug development process:

Source: Tufts Center for the Study of Drug Development

*Variations for biopharmaceuticals (e.g., “biologicals” = antibodies, cellular proteins):

Cost: $1.2 B for biotechnology product: $615 M, preclinical costs$626, clinical costs

Time: Longer development timeline8% longer to get through clinical testing and FDA review

Approval rate: 30% (1 of 3 biologicals that enter clinical testing) vs. 21% (1 of 5 pharmaceuticals that enter clinical testing)

*http://csdd.tufts.edu/NewsEvents/NewsArticle.asp?newsid=69Nov. 2006


5,000–10,000Screened

250 Enter Preclinical

Testing

5 Enter Clinical Testing

1Approved by the FDA

Compound Success Rates by Stage

Discovery:Literature, synthesis, lab screening;2-10 years

PreclinicalDevelopmentLab & animaltesting; 4-5 yrs

Phase 1: 1.5 yrsPhase II: 2 yrs

Phase III: 4-5 yrs

FDA Review & Approval: 1.5 yrsPost-Marketing

Testing

Timeline for Drug

Development

Avg of 15 yrs

File: Drug devel process-2.doc NWABR Jan. 2008

Drug Development ProcessScience – Regulations – Business Decisions

Chemistry:Making the drug Pre-clinical Testing Clinical Testing Marketing

Dosage Adverse effects Regulatory approval – FDA Recovery Fertility damage Carcinogenicity Formulation development

$0.8-1.2 B>500,000 10-15 yearsCompounds 1 Drug


File: Safety and Quality Tests.doc NWABRAug. 2008

Safety and Quality Tests

Test Purpose Name

Skin PenetrationOr Absorption Franz Cell

The Franz Cell is a vertical diffusion chamber that is used to determine the rate and extent of penetration of a test compound through the skin. Skin is positioned with a donor chamber above and a receptor chamber below. The skin may be from Yorkshire pigs or fresh or frozen human skin (from elective surgeries or cadavers). The test compound is applied to the top surface of the skin, while the skin’s dermis is in contact with a fluid, at body temperature, in the lower, receptor chamber. The skin may be covered, or not covered. After a pre-determined amount of time, the test is stopped and the amount of test compound is determined at the skin’s surface, within particular layers of the skin, and in the solution in the receptor chamber.

Allergy / Irritation Local Lymph Node Assay (LLNA)

The Local Lymph Node Assay (LLNA) uses mice to test for sensitization (allergic) potential, replacing a previous test in guinea pigs that required more animals. (Pre-test: Various concentrations of the test compound are applied to the ear to determine a concentration that will not cause irritation/swelling.) Concentrations of test compound that do not cause irritation are applied to the ears of mice once a day for three days, then on day 6, the mice are injected with a radioactive label (BrdU = bromodeoxyuridine). If the test compound causes sensitization (activation of immune system cells), there will be lymphocyte proliferation seen in the lymph nodes that drain the ears. The dividing lymphocytes will incorporate BrdU into their DNA. The lymph nodes are removed and analyzed for the number and type of cells incorporating BrdU. The test requires 20-25 mice.

Eye Irritation, a. Bovine Corneal Opacity and Permeability (BCOP) testCorrosion b. Isolated Chicken Eye (ICE) test

c. Draize eye test

http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox.htmICCVAM = Interagency Coordinating Committee on the Validation of Alternative Methods;responsible for testing the accuracy and reliability of test protocols.

June 2008: ICCVAM approved the BCOP Test Method and the ICE Test Method for use in a tiered testing, screening strategy to determine ocular hazards, with specific limitations for certain chemical classes and/or physical properties. Substances that test positive in these assays can be classified as ocular corrosives or severe irritants without further testing in animals. A substance that tests negative in the in vitro ocular toxicity test would need to be tested in the in vivo ocular test to identify possible in vitro false negatives and to identify moderate and mild ocular irritants.


http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox.htm


a. Bovine Corneal Opacity and Permeability (BCOP) testThis test measures the effect of a compound on the opacity and permeability of an isolated bovine cornea.PROTOCOL: Eyes from young adult cattle from slaughterhouses are kept at 4 degrees C and used within 5 hours after collection. Undamaged corneas are removed and put into O-ring holders with chambers containing saline solution bathing the front and back of the cornea. Corneas are held at 32 degrees C. for one hour. (a) The opacity of the cornea is measured. The saline is removed from the chamber next to the front of the cornea, the test compound is added to the chamber and gently rocked for 10 minutes; the test compound is removed by rinsing three times with saline and the opacity is measured. (b) Permeability of the cornea is determined by sodium-fluorescein staining for 90 minutes in the chamber on the front of the cornea, then removing the saline solution from the chamber on the under side of the cornea and measuring the amount of fluorescein dye present. (c) The corneas are fixed in a formalin solution for later histology if needed. The outcomes of the test are used to predict the in vivo ocular irritation potential.

b. Isolated Chicken Eye (ICE) testThis test measures the effect of a compound on corneal swelling, opacity, permeability (fluorescein dye retention), and morphological damage to the corneal surface on a chicken eye in an isolated system.PROTOCOL: Chicken heads from spring chickens, approximately 7 weeks old, male or female, with a weight range of 2.5-3.0 kg, are received from poultry slaughterhouses within two hours after humane killing. Eye lids are removed, corneas are stained with fluorescien for 10-20 seconds, rinsed, and microscopy is used to ensure the cornea is not damaged. Eyes are dissected from the eye socket with a visible portion of optic nerve remaining attached. The eyes are vertically mounted on a clamp in a superfusion chamber. Saline solution at 32 degrees C is pumped over the cornea. The corneas are examined for damage and corneal thickness is measured with a slit-lamp microscope.

For compound testing, the eye is removed from the clamp so that the cornea is horizontal. The test compound is applied and left for 10 seconds. The cornea is rinsed with saline and remounted in the superfusion chamber. The eye is examined at 30, 75, 120, 180, and 240 minutes (4 hrs) after treatment. At each time point, corneal opacity, corneal thickness, and morphological effects are evaluated. Fluorescein retention is examined at 30 minutes only. (Morphological effects include pitting, loosening of the epithelium, roughening of the corneal surface, and sticking of test compound to the corneal surface.) Photographs are recommended.

After the final time point, the eye is preserved in formaldehyde for possible later examination (histopathology).

Two additional ocular irritancy tests are being validated by ICCVAM:Isolated Rabbit Eye (IRE) testHen’s Egg Test – Chorioallantoic Membrane (HET-CAM)

c. The Draize eye test This test involves placing drops of a test compound in one eye of a rabbit and 24 hours later examining it for reddening, blistering, corneal clouding or corrosion. The healing can be observed after exposure. The original Draize test has been modified. Compounds known to be corrosive or irritating are not tested in the Draize test and anesthetics are used whenever possible.



Skin Corrosion, a. EPISKIN TM

Irritation b. Transcutaneous Electrical Resistance (TER) Assayc. Corrositex ®

http://iccvam.niehs.nih.gov/methods/dermal/dermal.htmhttp://ecvam.jrc.cec.eu.int/index.htm

European Centre for the Validation of Alternative Methods (ECVAM) validated and approved (April 2007) the use of EPISKIN as a full replacement for animal testing for assessing skin irritancy.

a. EPISKIN™ (from L’Oreal) is a three-dimensional human skin model comprised of a reconstructed epidermis and a functional stratum corneum. The test material is topically applied to the skin for 15 minutes with subsequent assessment of the effects on cell viability after 42 hrs using the colorimetric MTT assay. Release of IL-1a is a recommended adjunct to the MTT assay to increase the sensitivity of the test.

o www.iivs.org/images/EPISKIN_statement.pdf

b. The transcutaneous electrical resistance (TER) assay uses rat skin to identify corrosive materials by the ability to produce a loss of normal stratum corneum integrity and barrier function, which is measured as a reduction of the inherent transcutaneous electrical resistance below a predetermined threshold level.

Not approved as a full replacement test, but useable in a tier-testing strategy.c. Corrositex® is based on the ability of a corrosive chemical or chemical mixture to pass

through a biobarrier, by diffusion and/or destruction/erosion, and to elicit a color change in the underlying liquid Chemical Detection System (CDS). Corrositex® is useful as a stand-alone assay for evaluating the corrosivity or noncorrosivity of acids, bases, and acid derivatives. For other product classes, Corrositex® may be used as part of a tiered assessment strategy.

Moisture Evaporation Transepidermal Water Loss (TEWL)

One example: Delfin Technologies VapoMeter for measuring water evaporation. It is a closed chamber system that calculates the evaporation rate from the increase of relative humidity in the measurement chamber, compared to ambient humidity. http://www.delfintech.com/prod_vapo_faq.html

Mutagenesis/ Carcinogenesis Ames Test

The Ames test makes use of bacterial strains to determine if a compound causes mutations, which theoretically could lead to cancer in humans. Special strains of Salmonella typhimurium are used that require a mutation in order to grow into colonies on specific growth media. The special bacteria require different types of mutations (frameshift and point) in order to grow. The bacterial strains are grown in the presence of test substance. Only if mutations are induced will bacteria


http://www.delfintech.com/prod_vapo_faq.html

http://ecvam.jrc.cec.eu.int/index.htm

http://iccvam.niehs.nih.gov/methods/dermal/dermal.htm


grow into visible colonies. The tests may also be done in the presence of rat liver extract to simulate in vivo metabolism of the compound, since some compounds are mutagenic only after

being metabolized. The greater the number of colonies, the greater the mutagenic potential of the test compound.

Fever induction a. Limulus amebocyte lysate (LAL) testb. Cytokine Production

a. The Limulus amebocyte lysate (LAL) test; http://www.fda.gov/CBER/gdlns/lal.pdfThe LAL has been adopted to replace injections in rabbits to determine if a fever is produced in 24 hrs. It is required for medical devices and injected compounds that come in contact with blood or cerebral spinal fluid. The hemolymph (essentially, the blood cells) of the horseshoe crab react to bacterial endotoxins, lipopolysaccharide, and (1,3)-β-D-glucan. These are the same compounds that cause inflammation (and fever) in humans.

LAL is an aqueous extract of the blood cells of horseshoe crabs which forms a clot, becomes turbid, or changes color, depending on the technique, in the presence of bacterial endotoxin. The test sample is compared to a standard series of endotoxin dilutions. The endpoint or reaction times of these dilutions are used to calculate the amount of endotoxin present in the sample. All tests are performed in at least duplicate. Before performing the assay, one must determine if there is anything in the test sample/solution that will interfere (inhibit or enhance) the LAL assay.

b. Cytokine ProductionHuman blood can be used to determine whether or not a test compound causes white blood cells (leukocytes) to release cytokines (particular proteins) that are capable of signaling the initiation of a fever. A variety of commercial testing kits are available for quantifying multiple cytokines at once through colorimetric assays. Dilutions of the test compound are assayed and the results are quantified by comparison to known dilutions of cytokines.


File: Animal alternatives NYTimes.doc NWABRJan. 2008

Northwest Association for Resources (R)3.7 Biomedical Research


The New York Times, November 20, 2007http://www.nytimes.com/2007/11/20/business/worldbusiness/20cosmetics.html?_r=2&oref=slogin&pagewanted=print&oref=slogin

A New Science, at First Blush By DOREEN CARVAJAL

GRASSE, France — The delicate hybrids thriving in the balmy climes of Provence, southern France’s traditional perfume region, include sweet jasmine, May roses — and fresh layers of artificial human skin.

Scientists here are working feverishly to develop new technologies to test cosmetics before a European Union ban on animal testing begins in March 2009.These advanced materials — including reconstructed eye tissue and tiny circles of skin developed from donor cells harvested from cosmetic operations — are a vital part of the industry’s future as it faces rapidly tightening European regulations, rules that apply to any company wishing to sell in the 27-nation European Union.

The looming European ban is not only forcing multinational companies to adopt new practices. It is also bringing together regulators in Brussels with agencies from the world’s other large cosmetics markets — the Food and Drug Administration in the United States and the Ministry of Health in Japan — to harmonize regulation.Even more surprising, the new standards are pushing longtime secretive rivals to cooperate, grudgingly and sometimes with prodding from regulators and politicians.

The European commissioner for science, Janez Potocnik, appeared this month at a meeting for multinational companies and chided them for slowing the search for alternatives by failing to share information.

The stakes are high: Europe is the world’s leading cosmetics market, and it also exports more than $23.4 billion worth of cosmetics every year. Cosmetics exported from the United States to Europe amount to nearly $2 billion a year, about 7 percent of the European market. After the United States, Japan is the second leading provider of cosmetics to Europe .“Without question these regulations are having an impact,” said Dr. Alan Goldberg, director of the Center for Alternatives to Animal Testing at Johns Hopkins University in Baltimore. “What company is going to want to eliminate 450 million customers by not complying?”

The cosmetics giant L’Oréal has devoted more than $800 million in the last 20 years to the development of alternatives to animal testing, while its American rival, Procter & Gamble, maker of the Cover Girl line, has spent almost $225 million.“For the cosmetics industry, it’s a race,” said Hervé Groux, 45, a French immunology scientist who presides over a year-old research lab in Grasse that aids smaller companies lacking the resources of titans like L’Oréal and Procter & Gamble. “The rules are pushing everyone to move faster and to put more money into research.”

The European Commission itself is spending almost 25 million euros ($36.5 million) yearly on the search for animal alternatives, while many countries are seeding programs with annual budgets of 15 million to 20 million euros.

Mr. Groux’s lab, Immunosearch, had its official debut party Wednesday in a boxy industrial park, where Mr. Groux and his wife, a molecular biologist, and other newly recruited veteran researchers are striving to shape a new world of beauty research — and at the same time spare the lives of thousands of rabbits, mice, rats and guinea pigs.

As the 2009 deadline approaches, European regulators issue periodic tallies of the number of laboratory animals potentially spared by alternatives to animal tests, across all kinds of industrial uses. Part of the pressure for alternatives also stems from additional legislation, known as Reach, requiring companies to develop safety data on 30,000 chemicals over the next 11 years — research that could raise the prospect of increased animal testing.

In fact, the actual number of animals tested for cosmetics is small compared with medical or educational uses, according to a new European Commission report. But from 2002 to 2005 the tally grew 50 percent in Europe, to 5,571 animals.Much of that testing was taking place here in France, the country that leads Europe in testing and vigorously fought the ban, ultimately appealing, in vain, to the European Court of Justice.But it is also in Provence — a region fabled for its fragrances and the professional “noses” who create them — where scientists are gathering to work on alternative testing research in vitro, literally “in the glass.”


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In nearby Nice, SkinEthic, a 15-year-old company, is developing and manufacturing a line of cellular tools that includes a wide range of human tissues. Last year, SkinEthic was purchased by L’Oréal, which propelled the parent company into a dominant position in the testing field, with two critical patents on reconstructed skin. SkinEthic produces its own form of reconstructed skin, RHE, while L’Oréal holds the patent to Episkin, which its scientists developed in Lyon.

Episkin was validated this year by European regulators as a test tool that could fully replace animals. Its closest competitor, EpiDerm — developed by MatTek, a company in Ashland, Mass. — received only qualified approval for research use because the artificial skin reacted too sensitively, producing different results than natural skin would.

To make Episkin, donor keratinocyte cells, collected after breast and abdominal plastic surgery, are cultured in tiny wells of collagen gel, immersed in water, amino acids and sugars, and then air-dried for 10 days or aged to mimic mature skin by exposure to ultraviolet light.

Cosmetics are tested by smothering the almost babylike skin with the cosmetic material. The skin is checked for dying cells by adding a yellow chemical, MTT, which turns blue against living tissue, and then checked again for irritation.“We have finally succeeded in showing that artificial skin can fully replace rabbit testing,” said Thomas Hartung, the head of the European Center for the Validation of Alternative Methods in Italy.

His agency, which is part of the European Commission, is also in the midst of evaluating 12 methods for testing eye irritation, to replace the classic Draize test on rabbits that dates back to the mid-1940s.“If there’s a holy grail that we’re searching for, it’s a test for eye irritation,” said Mr. Hartung. “The issue has a very strong emotional factor.”

Other alternatives include using tiny membranes within chicken eggs for testing chemicals, because the blood vessels mimic human eye membranes. The center has also approved the use of cow and chicken eyeballs cast off from slaughterhouses.The mighty research budgets of the large multinational companies threaten to sweep aside smaller players with limited resources and different needs. But out of necessity, historic rivals in the secretive fragrance industry have joined to back Immunosearch, their homegrown research project, as a “deterrent force” to L’Oréal and P.& G.

Robertet, founded in 1850, and Mane Fils, dating back to the late 19th century, are supporting Immunosearch’s ambition of adapting L’Oréal’s Episkin to be more suitable for testing natural ingredients. Immunosearch is also benefiting from millions of dollars in private investments and government grants, as well as alliances with France’s National Science Research Center, nearby, and its National Institute for Research in Computer Science.

Robertet and Mane Fils, perfume companies run for four generations by rival families, acted out of self-interest. Both said they were alarmed that European regulations would force them to submit complex natural ingredients, like lavender essence, to the same tests intended for chemicals.

“Our industry has poorly defended natural ingredients because our essences have been classified as chemicals,” said Philippe Maubert, president of Robertet. “It’s unfortunate because these ingredients have existed for centuries.”

The companies worry that costly new European testing regulations could spell the end of many essential oils used in perfumery because the substances are a blend rising out of a distillation process that could fail existing chemical tests for safety.“Those tests were developed to make tests on pure substances with few impurities,” said Eric Angelini, the regulatory affairs manager at Mane. “But our essential oils are a natural blend of materials coming from the plant that is part of the distillation process.”

At the Champagne party on Wednesday to celebrate Immunosearch’s debut, city and regional officials crowded into the pristine laboratory, where kits of artificial skin layers that are sold by the dozen were stocked in a special machine warmed to human body temperature.

Jean-Pierre Leleu, the mayor of Grasse, toasted the future with a reminder that the industry has come full circle. In the 16th century, Grasse was a leather-tanning town that specialized in perfumed gloves. In the 18th century, the glove makers and perfumers split from the tanners to concentrate on perfumery.Now, Mr. Leleu said, the industry has turned its attention back to skin, albeit human.


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