June 2010 LANDSIEDEL 1

Chemical Industries Role in Tomorrows Toxicity Testing

Robert Landsiedel, Susanne Kolle, Tzutzuy Ramirez, Hennicke Kamp and Ben van Ravenzwaay

The Future of Chemical Toxicity Testing in the United States and Beyond

June 21st 2010

National Press Club, Washington DC Holeman Lounge, 13th Floor

Endocrine

Disruption

fog

Organ Toxicity

sand bar

Genotoxicity

fog

Developmental

Toxicity

shoal

Neurotoxicit

y sand bar

June 2010 LANDSIEDEL 3

Animal Welfare

TestingAcceptance

Validity

Time

Coststest substance

use

Producer’sResponsibility

REACH

CosmeticsDirective

C&L

non-EUlegislation

Alternative Methods Optimizing Toxicity Testing

June 2010 LANDSIEDEL 4

Acute Oral ToxicityCytotoxicity vs. Rat Acute Oral Toxicity

Assay:

Balb/c 3T3 mouse cell line

Viability assay using Neutral Red Uptake

Prediction model based on correlation of acute oral rat LD50 and cytotoxicity IC50

Data set:

203 pairs of cytotoxicity data and acute oral data (screening or OECD 423)

agrochemical active ingredients, agrochemical formulations, chemicals

June 2010 LANDSIEDEL 5

300 - 2000 mg/kg(cat. 4)

Acute Oral ToxicityCytotoxicity vs. Rat Acute Oral Toxicity

LD50 (acute oral rat) vs. EC50 Balb/c NRU, interpol. LD50

50300

2000

LD50 (GHS classification)

EC

50 I

nte

rpo

l. L

D5

0 [

mg

/kg

]

> 2000 mg/kg (cat. 5)

> 50 - 300 mg/kg(cat. 3)

≤ 50 mg/kg(cat. 2)

Overall concordance: 35 %Periodically assess the performance of methodsin routine testing.

June 2010 LANDSIEDEL 6

CPRG

CPRyeast ß-gal

ß-gal

estrogenic activity antiestrogenic activity

Yeast Estrogen/ Androgen Screening Assays (YES/YAS)

Recombinant yeast strains (human receptors and reporter gene)

Agonistic and antagonistic effects measureable

Determination of effect by reporter assay

Incubation of yeast cells with test substances

Measurement of hormonal activity and cytotoxicity

Reproductive Toxicity Endocrine Disruption

June 2010 LANDSIEDEL 7

YES/YASnegative

further development

positive

alternative structures

positivefurther development, but

develop alternative structures

focus in vivo testing

extended 28 day study Hershberger assay Uterotrophic assay early Reprotox testing

Reproductive ToxicityScreening Strategy

negative

June 2010 LANDSIEDEL 8

Mode of ActionMetabolomics

Test substance 28 day Rat studies Metabolite profile in

plasma

MetaMap®TOX

identification of relevant metabolites correlation analysis against

reference profiles comparison against specific patterns

for toxicity

Identification of target organs modes of action substance classes

June 2010 LANDSIEDEL 9

Three patterns of liver effects:rats (females left and males right) treated with liver enzyme inducers, liver toxic compounds and peroxisome proliferatorsrelative to untreated controls

Metabolomics MoA Identification

June 2010 LANDSIEDEL 10

Metabolomics Example: 2- and 4-Acetylaminofluorene

2-Acetylaminofluorene

- strong liver enzyme inducer- liver carcinogen

- immune suppressant- bladder carcinogen

4-Acetylaminofluorene

- slight liver enzyme inducer- no liver carcinogen

- lipid accumulation in liver- immune suppressant

NH

ONH

O

June 2010 LANDSIEDEL 11

Metabolite profile compared to other liver enzyme inducers2-AAF 4-AAF

Metabolomics Example: 2- and 4-Acetylaminofluorene

Extract more informationfrom (regulatory) studies,

to provide a sound scientific basis for grouping and read across of chemicals:

from QSAR to QBAR

June 2010 LANDSIEDEL 12

Acute dermal toxicity:

Total number of selective (relevant) classificationsbased on LD50 dermal in ECB data base: < 1%

Mouse cancer bioassay:

The mouse does not add significantly to conclusionson cancer risk assessment (compared to rat study)

90 day and 12 month dog studies:

LOEL in 90 day dog is lower than that in rats for ~50% of compounds

LOEL in 90 day dog study is more than 3-fold compared to 12 month study for about 90% of compounds

Redundancy in Animal Testing of Chemicals

HESI ACSA Task Force

June 2010 LANDSIEDEL 13

New paradigmCurrent paradigmAnimals

7681272Total

4872Dogs

0520Mice

720680Rats

Potential reduction in Animal Testing for Systemic Toxicity

HESI ACSA Task Force

Periodically assess the regulatory impact of studies,

and delete those that are not used.

June 2010 LANDSIEDEL 14

Endocrine

Disruption

Organ Toxicity

Genotoxicity

Developmental

Toxicity

Neurotoxicit

y

Vielen Dank.

Thank you.