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Regulatory Toxicology and Pharmacology 50 (2008) 322–335

Mutagenicity assessment of acrylate and methacrylate compoundsand implications for regulatory toxicology requirements

F.R. Johannsen a, Barbara Vogt b,*, Maureen Waite c, Randy Deskin d

a FRJ-TOX, L.L.C., 12346 Coppersmith Court, St. Louis, MO 63131, USAb Cytec Industries, 1937 West Main Street, Stamford, CT 06904, USA

c Cytec Industries, 1950 Lake Park Drive, Smyrna, GA 30080, USAd Cytec Industries, 5 Garret Mountain Plaza, West Paterson, NJ 07424, USA

Received 3 February 2007Available online 1 February 2008

Abstract

Esters of acrylic acid and methacrylic acid, more commonly known as acrylates and methacrylates, respectively, are key raw materialsin the coatings and printing industry, with several of its chemical class used in food packaging. The results of over 200 short-term in vitro

and in vivo mutagenicity studies available in the open literature have been evaluated. Despite differences in acrylate or methacrylate func-tionality or in the number of functional groups, a consistent pattern of test response was seen in a typical regulatory battery of muta-genicity tests. No evidence of point mutations was observed when acrylic acid or over 60 acrylates and methacrylates were investigated inSalmonella bacterial tests or in hprt mutation tests mammalian cells, and no evidence of a mutagenic effect was seen when tested in wholeanimal clastogenicity and/or aneuploidy (chromosomal aberration/micronucleus) studies. Consistent with the in vivo testing results,acrylic acid exhibited no evidence of carcinogenicity in chronic rodent cancer bioassays. In contrast, acrylic acid and the entire acrylateand methacrylate chemical class produced a consistently positive response when tested in the mouse lymphoma assay and/or otherin vitro mammalian cell assays designed to detect clastogenicity. The biological relevance of this in vitro response is questioned basedon the non-concordance of in vitro results with those of in vivo studies addressing the same mutagenic endpoint (clastogenicity). Thus,in short-term mutagenicity tests, the acrylates and methacrylates behave as a single chemical category, and genotoxicity behavior of asimilar chemical can be predicted with confidence by inclusion within this chemical class, thus avoiding unnecessary testing.� 2008 Elsevier Inc. All rights reserved.

Keywords: Acrylate; Methacrylate; Mutagenicity; Genotoxicity; Regulatory test batteries; Chemical categories; Structure activity; SAR; Mouse lymph-oma assay; Mouse micronucleus assay; Bacterial reverse mutation assay; Carcinogenicity

1. Introduction

Acrylates and methacrylates are key raw materials usedin diverse applications, including coatings, printing inks,varnishes, sealants, caulks, adhesives, textiles and plastics,and as chemical intermediates. Acrylates are esters ofacrylic acid, resulting from a catalyzed condensationbetween one acrylic acid moiety (monofunctional) or morethan one acrylic acid moiety (multifunctional) with an alco-hol. Methacrylates are esters formed in a similar manner

0273-2300/$ - see front matter � 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.yrtph.2008.01.009

* Corresponding author. Fax: +1 203 321 2978.E-mail address: [email protected] (B. Vogt).

using methacrylic acid. Both the mono- and multifunc-tional acrylate analogs and the methacrylate esters (mono-functional and multifunctional) possess a similar spectrumand pattern of toxicity (Moss et al., 1997).

The diversity of industrial applications of acrylates andmethacrylates requires that they must meet a variety of reg-ulatory mutagenicity testing requirements (Table 1).Screening mutagenicity test requirements, for example,for both the U.S. EPA (Environmental Protection Agency)HPV (High Productive Volume) Challenge program andthe broader OECD (Organization of Economic and Coop-erative Development) HPV program consist of a 2-testin vitro mutagenicity battery, which includes: a gene reverse

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Table 1Mutagenicity requirements for selected regulatory programs in the United States and the European Union

In vitro In vivo References

Bacterialmutagenicity

Mammalianmutagenicity

Chromosomalaberrations

Erythrocyte micronucleusor bone marrowchromosomal aberration

OECD SIDS/HPVp

O O C OECD (2005)U.S. EPA/HPV SIDS/HPV

p pO Cimino (2006)

U.S. EPA New Chemicalsp p

Cimino (2006)U.S. FDA Food Contact

Notificationa

pO O C CFSAN (2002)

EU Dangerous SubstancesDirective, VII A

Registrationp p

C European Commission(1992)

EU EFSA Food Contacta p p pC EFSA (2006)

pdenotes a requirement.

O denotes optional among other optional tests.C denotes conditional if previous in vitro tests are positive for mutagenic results.

a Conditional on levels of migration into food 650 ppb.

F.R. Johannsen et al. / Regulatory Toxicology and Pharmacology 50 (2008) 322–335 323

mutation assay in bacteria (Ames assay) and a chromo-somal aberration assay in cultured mammalian cells. Newchemical notifications in the European Union (EU) requirea battery of two mutagenicity studies. While most newchemicals do not require premanufacturing testing in theU.S., those chemicals meeting specified volume and expo-sure criteria or are included in specialized chemical catego-ries, require testing in a gene mutation assay and an in vivoclastogenicity assay (Cimino, 2006).

A number of these acrylates are used in food packagingmaterials and, as such, they must meet the requirements offood contact notifications around the world. The U.S.FDA (Food and Drug Administration) Center for FoodSafety and Applied Nutrition (CFSAN, 2002) recommendsa 2-test battery of mutagenicity studies for food contactsubstances with a cumulative estimated daily intake nogreater than 50 ppb in the diet. The European Food SafetyAuthority (EFSA) requires submission of a battery of threemutagenicity tests for materials migrating at levels less than50 ppb (EFSA, 2006a). If any single result from these testsis suggestive of mutation or chromosomal disruption, thenat least one in vivo mammalian genotoxicity assay isrequired, most commonly the mouse micronucleus assay.

Genotoxicity testing has been completed on many of theacrylates and methacrylates. In this paper, we present asummary of published mutagenicity results indicating thatthese compounds should be considered nonmutagenic inthe whole animal. The data clearly support the conceptof a category for mutagenicity behavior for acrylates andmethacrylates. Further, this data offers grounds for re-assessment of the multi-test battery recommended bynumerous regulatory agencies, insofar as this chemical cat-egory is concerned.

2. Materials and methods

A number of public literature sources have been investigated to iden-tify all available short-term genotoxicity studies conducted with acrylicacid, methacrylic acid, and members of the acrylic acid ester (acrylates)

and methacrylic acid ester (methacrylates) family of chemicals. Studieswere identified which used either of the acids or one of the monofunctionalor multifunctional (including di-, tri- and tetra-functional) derivatives ofthese two acids. The sphere of genotoxicity tests included in this reviewwere those most frequently used by global regulatory bodies for initialhazard screening purposes and tended to follow study designs consistentwith current regulatory guidelines. Study types included as part of thisreview were: Salmonella reverse mutation (Ames test), mouse lymphomaL5178Y tk± locus, mouse lymphoma hgprt gene assay, in vitro chromo-somal aberration, sister-chromatid exchange (SCE), Chinese Hamsterovary (CHO/hgprt), V79/hgprt, in vitro cell transformation, in vitro

unscheduled DNA synthesis (UDS), in vitro micronucleus, in vivo micro-nucleus, in vivo chromosomal aberration, and in vivo SCE . For in vitro

assays, multiple mammalian cell lines, primarily those of rodents andhumans, were considered; multiple animal species and routes of dosingwere included in evaluation of the in vivo studies. Where available, primaryliterature citations have been cited. Multiple findings in a similar test sys-tem by different researchers have been included to provide weight-of-the-evidence of study findings. In a number of cases, results of short-termgenotoxicity studies by parties involved in either the conduct or authoriza-tion of conduct of these studies were reported in secondary source docu-ments and have also been included in this review, where validity of thefindings was judged to be high. Examples of such studies came from (1)testing by the U.S. National Toxicology Program (NTP), the results ofwhich are listed on their official website or Technical Documents butnot specifically reported or (2) robust summary documentation of a studysubmitted for peer review under voluntary programs to share hazard dataon a national (EPA HPV Challenge program) or global (OECD/ICCAHPV program) basis, often resulting in a final, documented assessmentand (3) review documents summarizing the decisions of EFSA. In allcases, final conclusions reached in those studies were taken from thoseof the individual authors or peer-reviewed summary.

3. Results

A substantial number of monofunctional and multifunc-tional acrylates and methacrylates have been tested usingstandard test methodology currently found in internation-ally accepted mutagenicity testing guidelines (OECD,1997). As seen in Table 2, acrylic acid, methacrylic acidand the vast majority of acrylates and methacrylates thathave been tested are inactive in the bacterial reverse muta-tion assay, whether tested with or without metabolic acti-vation in a series of standard Salmonella tester strains.

Table 2Results of reverse mutation bacterial assay with acrylic and methacrylic acid and their monofunctional and multifunctional acid esters

Acrylic/methacrylic acid/ester CAS No. Test results Reference(s)

(I) Acrylic acid

Acrylic acid 79-10-7 (1) Inactive (1) Zeiger et al. (1987)(2) Inactive (2) Cameron et al. (1991)

(II) Monofunctional acrylates

Methyl acrylate 96-33-3 (1) Inactive (1) Waegenmachers and Bensink (1984)(2) Inactive (2) Zeiger et al. (1987)

Ethyl acrylate 140-88-5 (1) Inactive (1) Waegenmachers and Bensink (1984)(2) Inactive (2) Haworth et al. (1983)

2-Hydroxyethyl acrylate 818-61-1 Inactive Watanabe et al. (1996)n-Butyl acrylate 141-32-2 (1) Inactive (1) Waegenmachers and Bensink (1984)

(2) Inactive (2) Zeiger et al. (1987)iso-Butyl acrylate 106-63-8 Inactive Zeiger et al. (1987)tert-Butyl acrylate 1663-39-4 Inactive Waegenmachers and Bensink (1984)2-Ethylhexyl acrylate 103-11-7 Inactive Zeiger et al. (1987)Pentyl acrylate 2998-23-4 Inactive Waegenmachers and Bensink (1984)Neopentyl acrylate 4513-36-4 Inactive Waegenmachers and Bensink (1984)Hexyl acrylate 2499-95-8 Inactive Waegenmachers and Bensink (1984)iso-Octyl acrylate 29590-42-9 Inactive Gordon et al. (1991)

(III) Multifunctional acrylates

Glycerol propoxydiacrylate 103534-15-2 Inactive Andrews and Clary (1986)Pentaerythritol triacrylate 3524-68-3 (1) Inactive (1) Zeiger et al. (1987)

(2) Inactive (2) Andrews and Clary (1986)Triethyleneglycol diacrylate 1680-21-3 Inactive Andrews and Clary (1986)Tetraethyleneglycol diacrylate 17831-71-9 Inactive Andrews and Clary (1986)Dipropyleneglycol diacrylate 57472-68-1 Inactive Andrews and Clary (1986)Glycerol propoxytriacrylate 52408-84-1 Inactive Andrews and Clary (1986)Trimethylolpropane ethoxytriacrylate 28961-43-5 Inactive Andrews and Clary (1986)Butanediol diacrylate 1070-70-8 Inactive Waegenmachers and Bensink (1984)1,5-Pentanediyl diacrylate 36840-85-4 Inactive Waegenmachers and Bensink (1984)Ethyleneglycol diacrylate 2274-11-5 (1) Inactive (1) Waegenmachers and Bensink (1984)

(2) Inactive (2) Zeiger et al. (1987)Trimethylolpropane triacrylate 15625-89-5 (1) Inactive-rat S9 (1) Cameron et al. (1991)

(2) Positive-hamster S9 (2) Cameron et al. (1991)Hexanediol diacrylate 13048-33-4 (1) Inactive (1) Waegenmachers and Bensink (1984)

(2) Inactive (2) Andrews and Clary (1986)(3) Inactive (3) Zeiger et al. (1987)

Neopentanediol diacrylate 2223-82-7 (1) Inactive (1) Waegenmachers and Bensink (1984)(2) Inactive (2) Andrews and Clary (1986)

(IV) Methacrylic acid

Methacrylic acid 79-41-4 (1) Inactive (1) Haworth et al. (1983)(2) Inactive (2) Querens et al. (1981)

(V) Monofunctional methacrylates

Methyl methacrylate 80-62-6 (1) Inactive (1) Waegenmachers and Bensink (1984)(2) Inactive (2) Zeiger et al. (1987)(3) Inactive (3) Querens et al. (1981)(4) Inactive (4) Schweikl et al. (1998)

Ethyl methacrylate 97-63-2 (1) Inactive (1) Waegenmachers and Bensink (1984)(2) Inactive (2) Zeiger et al. (1987)

Hydroxyethyl methacrylate 868-77-9 (1) Inactive (1) Waegenmachers and Bensink (1984)(2) Inactive (2) Schweikl et al. (1998)

n-Propyl methacrylate 2210-28-8 Inactive Zeiger et al. (1987)iso-Propyl methacrylate 4655-34-9 Inactive Zeiger et al. (1987)Butyl methacrylate 97-88-1 (1) Inactive (1) Waegenmachers and Bensink (1984)

(2) Inactive (2) Zeiger et al. (1987)(3) Inactive (3) EPA/OTS (1986)

tert-Butyl methacrylate 585-07-9 Inactive Waegenmachers and Bensink (1984)iso-Butyl methacrylate 97-88-9 Inactive Zeiger et al. (1987)Pentyl methacrylate 2849-98-1 Inactive Waegenmachers and Bensink (1984)Neopentyl methacrylate 2397-76-4 Inactive Waegenmachers and Bensink (1984)Hexyl methacrylate 142-09-6 (1) Inactive (1) Waegenmachers and Bensink (1984)

(2) Inactive (2) Zeiger et al. (1987)Isodecyl methacrylate 29964-84-9 Inactive Zeiger et al. (1987)

324 F.R. Johannsen et al. / Regulatory Toxicology and Pharmacology 50 (2008) 322–335

Table 2 (continued)

Acrylic/methacrylic acid/ester CAS No. Test results Reference(s)

Dodecyl (Lauryl) methacrylate 142-90-5 Inactive Elf Atochem (1993)Octadecyl (Stearyl) methacrylate 32360-05-7 Inactive Elf Atochem (1994)Isobornyl methacrylate 7534-94-3 Inactive Rohm and Haas (1999)Octyl methacrylate 2157-01-9 Inactive Zeiger et al. (1987)Allyl methacrylate 96-05-9 Inactive Elf Atochem (1991)2-Hydroxypropyl methacrylate 27813-02-1 Inactive Zeiger (2000)Glycidyl methacrylate 106-91-2 (1) Positive (1) Querens et al. (1981)

(2) Positive (2) Schweikl et al. (1998)(3) Positive (3) EPA/OTS (1992)(4) Inactive (4) Waegenmachers and Bensink (1984)

(VI) Multifunctional methacrylates

1,2-Ethanediol dimethacrylate 97-90-5 Inactive Waegenmachers and Bensink (1984)1,4-Butanediol dimethacrylate 2082-81-7 Inactive Waegenmachers and Bensink (1984)1,5-Pentanediol dimethacrylate 13675-34-8 Inactive Waegenmachers and Bensink (1984)Neopentanediol dimethacrylate 1985-51-9 Inactive Waegenmachers and Bensink (1984)1,6-Hexanediol dimethacrylate 6606-59-3 Inactive Waegenmachers and Bensink (1984)Diethylene glycol dimethacrylate 2358-84-1 Inactive Waegenmachers and Bensink (1984)Urethane glycol dimethacrylate 28654-11-7 Inactive Schweikl et al. (1998)Bisphenol A-glycidyl dimethacrylate 72869-86-4 (1) Inactive (1) Querens et al. (1981)

(2) Inactive (2) Schweikl et al. (1998)Ethylenegylcol dimethacrylate 97-90-5 Inactive Cameron et al. (1991)Trimethylolpropane trimethacrylate 3290-92-4 (1) Inactive-rat S9 (1) Cameron et al. (1991)

(2) Positive-hamster S9 (2) Cameron et al. (1991)Triethyleneglycol dimethacrylate 109-16-0 Inactive Schweikl et al. (1998)


An exception is GMA (glycidyl methacrylate), derivedfrom glycidol which, as a member of the class of epoxides,is mutagenic in the bacterial reverse mutation assay(McCann et al., 1975). Positive responses reported withtrimethylolpropane triacrylate (TMPTA) and trimethylol-propane trimethacrylate (TMPTMA) occurred in a singleSalmonella tester strain (TA1535) only when a hamsterliver metabolic activation system was used. Both were inac-tive in all Salmonella strains without metabolic activationand in TA1535 when a conventional rat liver metabolicactivation system was used (NLM, 2006). While hamsterliver homogenates are used to assess chemicals containingthe azo moiety, no such structural alert is present in eitherof these chemicals. Thus, when compared to equivalentstudy designs used for other acrylates and methacrylates,TMPTA and TMPTMA should be considered inactive inthe bacterial reverse mutation assay.

Results of mouse lymphoma testing on acrylic acid andmonofunctional and multifunctional acrylates and methac-rylates are reported in Table 3. Acrylic acid produced apositive response with and without metabolic activation.The response observed was considered relatively weak, inthat less than tripling of the background mutation ratewas reported. With the exception of iso-octyl acrylate, allacrylates and methacrylates tested were positive in themouse lymphoma assay. With several of the acrylates, posi-tive responses were characterized as modest (2- to 3-fold)mutational increases which occurred at cytotoxic dose lev-els where the Relative Total Growth (RTG) was less than50% of control values.

A few short-term mammalian cell culture assays otherthan the mouse lymphoma assay have been used to identify

the capability of several acrylates and methacrylates to affectgene mutational events. Two such assays which measureeffects occurring at a particular gene (hgprt) locus have beenused and include the Chinese Hamster ovary (CHO)/hgprt

assay and the Chinese Hamster lung V79/hgprt assay. Testresults with acrylic acid and acrylates and methacrylatestested in these assays are summarized in Table 4. No evi-dence for induction of point mutations was observed inany of these tests in mammalian cell lines, although Schweikland Schmaltz (1999) identified chromosomal effects in thissystem with triethylene glycol dimethacrylate (TEGDMA)and glycidyl methacrylate (Schweikl et al., 1998).

In vitro tests designed to either directly or indirectly mea-sure chromosomal events involving chromosomal structureor number include the chromosomal aberration assay, usingeither Chinese Hamster ovary (CHO), Chinese hamsterlung cells (CHL), human lymphocytes (HL) or even ratbone marrow cell cultures. Formation of micronuclei (pre-sumably formed from broken or disjointed chromosomes)and a more indirect in vitro measure of chromosomal dam-age, the sister chromatid exchange (SCE) assay (signalinginterference with the reciprocal interchanges of the twochromatid arms within a single chromosome) have alsobeen used. Results of acrylic acid and acrylate and methac-rylate testing in mammalian cell assays designed to detectin vitro clastogenic potential are found in Table 5. Positivemutagenic responses were seen with acrylic acid itself andmost other mono-and multifunctional acrylates and meth-acrylates tested for chromosome damage.

The results of two other in vitro assays, capable ofbroadly measuring cellular events affecting DNA, havebeen reported in Table 6. The unscheduled DNA synthesis

Table 3Results of mouse lymphoma testing on acrylic acid, monofunctional and multifunctional acrylates and methacrylates

Acrylic/methacrylic acid/ester CAS No. Mouse lymphoma test results Reference(s)

(I) Acrylic acid

Acrylic acid 79-10-7 (1) Positive (1) Cameron et al. (1991)(2) Positive (2) Moore et al. (1988)


Methyl acrylate 96-33-3 (1) Positive (1) Moore et al. (1991)(2) Positive (2) Moore et al. (1988)

Ethyl acrylate 140-88-5 (1) Positive (1) Moore et al. (1991)(2) Positive (2) Moore et al. (1988)

2-Hydroxyethyl acrylate 818-61-1 Positive Dearfield et al. (1989)2-Ethylhexyl acrylate 103-11-7 Positive Dearfield et al. (1989)iso-Octyl acrylate 29590-42-9 Inactive Gordon et al. (1991)


Trimethylolpropane ethoxytriacrylate 28961-43-5 Positive Andrews and Clary (1986)Tripropyleneglycol diacrylate 42978-66-5 (1) Positive (1) Andrews and Clary (1986)

(2) Positive (2) Dearfield et al. (1989)Glycerol propoxydiacrylate 103534-15-2 Positive Andrews and Clary (1986)Pentaerythritol triacrylate 3524-68-3 (1) Positive (1) Dearfield et al. (1989)

(2) Positive (2) Andrews and Clary (1986)Triethyleneglycol diacrylate 1680-21-3 (1) Positive (1) Andrews and Clary (1986)

(2) Positive (2) Dearfield et al. (1989)(3) Positive (3) Moore et al. (1991)

Tetraethyleneglycol diacrylate 17831-71-9 Positive Andrews and Clary (1986)Ethyleneglycol diacrylate 2274-11-5 Positive Cameron et al. (1991)Trimethylolpropane triacrylate 15625-89-5 (1) Positive (1) Andrews and Clary (1986)

(2) Positive (2) Cameron et al. (1991)(3) Positive (3) Dearfield et al. (1989)

Hexanediol diacrylate 13048-33-4 Positive Andrews and Clary (1986)

(IV) Monofunctional methacrylates

Methyl methacrylate 80-62-6 (1) Positive (1) Moore et al. (1988)(2) Positive (2) Amtower et al. (1986)(3) Positive (3) Doerr et al. (1988)(4) Positive (4) Dearfield et al. (1991)

Ethyl methacrylate 97-63-2 Positive Moore et al. (1988)

(V) Multifunctional methacrylates

Trimethylolpropane trimethacrylate 3290-92-4 Positive Dearfield et al. (1989)Ethylenegylcol dimethacrylate 97-90-5 Positive Cameron et al. (1991)

Table 4Mutagenic response in mammalian cell culture assays for gene mutations other than the mouse lymphoma assay for acrylic acid and several acrylates andmethacrylates

Acrylic acid and esters CAS No. Tests Test results Reference(s)

(I) Acrylic acid

Acrylic acid 79-10-7 CHO/HGPRT Inactive Anon. (2002)

(II) Methyl acrylate

Monofunctional acrylates 96-33-3 CHO/HGPRT Inactive Moore et al. (1989)Ethyl acrylate 140-88-5 CHO/HGPRT Inactive Moore et al. (1989)2-Ethylhexyl acrylate 103-11-7 CHO/HGPRT Inactive Moore et al. (1991)


Neopentyl glycol diacrylate 2223-82-7 CHO Inactive EPA/OTS (1981)


Glycidyl methacrylate 106-91-2 V79/HGPRT Positive Schweikl et al. (1998)Methyl methacrylate 80-62-6 V79/HGPRT Inactive Schweikl et al. (1998)Hydroxyethyl methacrylate 868-77-9 V79/HGPRT Inactive Schweikl et al. (1998)


Triethyleneglycol dimethacrylate 109-16-0 V79/HGPRT Positive Schweikl et al. (1998)Bisphenol A-glycidyl dimethacrylate 72869-86-4 V79/HGPRT Inactive Schweikl et al. (1998)Urethane glycol dimethacrylate 28654-11-7 V79/HGPRT Inactive Schweikl et al. (1998)


Table 5Results of in vitro mammalian chromosomal aberration, sister-chromatid exchanges or micronucleus tests with acrylic acid and several acrylates andmethacrylates


(I) Acrylic acid

Acrylic acid 79-10-7 (1) Chromosomal Aberrations. (1) Positive (1) Anon. (2002)(2) In vitro micronucleus (2) Inactive (2) Wiegand et al. (1989)


Methyl acrylate 96-33-3 (1) CHL Chromosomal Aberrations. (1) Positive (1) Ishidate et al. (1984)(2) CHO Chromosomal Aberrations. (2) Positive (2) Moore et al. (1991)(3) CHO Chromosomal Aberrations. (3) Positive (3) Doerr et al. (1988)(4) CHO Chromosomal Aberrations. (4) Positive (4) Ishidate et al. (1984)(5) CHO Chromosomal Aberrations. (5) Positive (5) Parker et al. (1988)(6) CHO Chromosomal Aberrations. (6) Positive (6) Oberly et al. (1993)

Ethyl acrylate 140-88-5 (1) CHL Chromosomal Aberrations. (1) Positive (1) Ishidate et al. (1984)(2) Yeast Chromosomal Aberrations. (2) Positive (2) Zimmerman and Mohr (1992)(3) CHO Chromosomal Aberrations. (3) Positive (3) Moore et al. (1991)(4) CHO Chrom. Ab. (4) Positive (4) Doerr et al. (1988)(5) CHO Chrom. Ab. (5) Positive (5) Loveday et al. (1990)(6) SCE (6) Inactive (6) Anon. (2002)

n-Butyl acrylate 141-32-2 (1) In vitro micronucleus (1) Inactive (1) Ishidate et al. (1984)(2) SHE Chromosomal Aberrations. (2) Inactive (2) Wiegand et al. (1989)

2-Ethylhexyl acrylate 103-11-7 (1) CHO Chromosomal Aberrations. (1) Positive (1) Moore et al. (1991)(2) CHO Chromosomal Aberrations. (2) Positive (2) Parker et al. (1988)(2) SCE (3) Positive (3) Rohm and Haas (1982)

2-Hydroxypropyl acrylate 999-61-1 CHO Chromosomal Aberrations. Positive EPA/OTS (1988)


Neopentyl glycol diacrylate 2223-82-7 (1) CHO Chromosomal Aberrations. (1) Inactive (1) EPA/OTS (1981)(2) SCE (2) Inactive (2) EPA/OTS (1981)

Triethyleneglycol diacrylate 1680-21-3 (1) CHO Chromosomal Aberrations. (1) Positive (1) Moore et al. (1991)(2) CHO Chromosomal Aberrations. (2) Positive (2) Parker et al. (1988)

Trimethylolpropane triacrylate 15625-89-5

(1) CHO Chromosomal Aberrations. (1) Positive (1) Moore et al. (1991)(2) CHO Chrom. Ab. (2) Positive (2) Parker et al. (1988)(3) Human lymphocyte-Chromosomal Aberrations. (3) Positive (3) ACC (2005a)


Methyl methacrylate 80-62-6 (1) CHO Chromosomal Aberrations. (1) Positive (1) Bigatti et al. (1989)(2) SCE (2) Positive (2) NLM (2006)

2-Hydroxyethyl methacrylate 868-77-9 CHL Chromosomal Aberrations. Positive MHW-Japan (1997)Allyl methacrylate 96-05-9 Human lymphocyte Chromosomal Aberrations. Inactive Elf Atochem (1991)Glycidyl methacrylate 106-91-2 CHL Chromosomal Aberrations. Positive MHW-Japan (1997)


Ethylenegylcol dimethacrylate 97-90-5 Chromosomal Aberrations. Positive ICI (2001)Dicyclopentenyloxyethyl methacrylate 68586-19-

6(1) CHO Chromosomal Aberrations. (1) Positive (1) Moore et al. (1991)(2) CHO Chromosomal Aberrations. (2) Positive (2) Parker et al. (1988)

Triethyleneglycol dimethacrylate 109-16-0 V79 micronuclei Positive Schweikl et al. (1998)

Table 6Results of additional gene toxicity in vitro assays with acrylic acid and acrylic and methacrylic acid esters


(I) Acrylic acid

Acrylic acid 79-10-7 (1) UDS (1) Inactive (1) Wiegand et al. (1989)(2) UDS (2) Inactive (2) Anon. (2002)(3) Cell transformation (3) Inactive (3) Wiegand et al. (1989)


n-Butyl acrylate 141-32-2 (1) Cell transformation (1) Inactive (1) NTP (1986)(2) UDS (2) Inactive (2) Wiegand et al. (1989)

2-Ethylhexyl acrylate 103-11-7 (1) UDS (1) Equivocal (1) Slesinski (1980)(2) Cell transformation (2) Inactive (2) Rohm and Haas (1982)

iso-Octyl acrylate 29590-42-9 Cell transformation Inactive Gordon et al. (1991)


Neopentyl glycol diacrylate 2223-82-7 UDS Inactive (1) EPA/OTS (1981)



assay in cultured mammalian cells is capable of measuringrepair of DNA damage induced by a number of chemicals.The cell transformation assay, also conducted in mamma-lian cells, is designed to identify the earliest recognizablephenotype in the multi-step transformation process leadingto neoplasia. For the few mono- and multifunctional acry-lates tested in these assays, a consistent pattern of non-response was reported and results proved nongenotoxic(inactive) or equivocal.

Table 7 contains results of in vivo tests, either rat ormouse cytogenetics or mouse micronucleus assays whichhave been conducted with 18 acrylate and methacrylateesters and acrylic acid. Whether testing by the oral, dermalor inhalation exposure routes, no mutagenic response hasbeen observed with acrylic acid or any of the mono- ormultifunctional acrylates tested. With the exception of

Table 7Results of in vivo chromosomal aberration/micronucleus tests with acrylic aci

Acrylate CAS No. Test system

(I) Acrylic acid

Acrylic acid 79-10-7 Chromosomal Aberrations.—o


Ethyl acrylate (EA) 140-88-5 (1) Micronucleus-IP

(2) Micronucleus—dermal(3) SCE-IP(4) Chromosomal Aberrations.

2-Ethylhexyl acrylate 103-11-7 Chromosomal Aberrations.—on-Butyl acrylate 141-32-2 (1) Chromosomal Aberrations.iso-Butyl acrylate 106-63-8 Micronucleus-IPMethyl acrylate 96-33-3 (1) Micronucleus-IP

(2) Micronucleus—Oral(3) Micronucleus—Inhalation


Trimethylolpropane triacrylate 15625-89-5 Micronucleus—oralTripropyleneglycol diacrylate 42978-66-5 Micronucleus—dermalPentaerythritol triacrylate 3524-68-3 Micronucleus-mouse—dermal


Methyl methacrylate 80-62-6 (1) Rat Chromosomal AberratInhal.(2) Mouse micronucleus—oral(3) Mouse micronucleus—oral

Ethyl methacrylate 97-63-2 Mouse micronucleus—oralHydroxypropyl methacrylate 27813-02-1 Mouse micronucleus—oraln-Butyl methacrylate 97-88-1 Mouse micronucleus—oraliso-Butyl methacrylate 97-88-9 Mouse micronucleus—oralGlycidyl methacrylate 106-91-2 (1) Mouse micronucleus—oral

(2) Mouse micronucleus-IPDodecyl (Lauryl) methacrylate 142-90-5 Mouse micronucleus—oralOctadecyl (Stearyl)

methacrylate32360-05-7 Mouse micronucleus—oral

(IV) Multiufunctional methacrylates

Trimethylolpropanetrimethacrylate

3290-92-4 Mouse micronucleus—route un

glycidyl methacrylate, which gave inconsistent responseswhen tested by two different dosing routes (IP and oral),the remaining mono- and multifunctional methacrylateswere also inactive.

4. Discussion

With few exceptions, the acrylates and methacrylatesreviewed are nonmutagenic in point mutation tests. Thetest results appear remarkably consistent within each ofseveral types of tests across the functional spectrum ofacrylates and methacrylates, with no apparent differencesin response related to structure (number of acrylate ormethacrylate moieties present, or formation from theirbase acids). GMA, which induced mutations in the bacte-rial reverse mutation assay, is derived from a chemical pre-

d and acrylic and methacrylic acid esters

Results Reference(s)

ral Inactive McCarthy et al. (1992)

(1) Inactive-weight-of-the-evidence(irreproduciblein other studies)

(1a) Przybojewska et al. (1984)(positive)

(1b) Ashby et al. (1989) (inactive)(1c) Kligerman et al. (1981) (inactive)

(2) Inactive (2) Tice et al. (1997)(3) Inactive (3) Kligerman et al. (1981)

-IP (4) Inactive (4)Kligerman et al. (1981)ral Inactive Anon. (2002)- Inhal. (1) Inactive (1) Engelhardt and Klimisch (1983)

Inactive OECD (2005)(1) ‘‘Positive” (seeEA)

(1) Przybojewska et al. (1984)

(2) Inactive (2) Hachitani et al. (1981)(3) Inactive (3) Sofuni et al. (1984)

Inactive ACC (2005b)Inactive Tice et al. (1997)Inactive NTP (2007)

ions.- (1) Inactive (1) ICI (2002)

(2) Inactive (2) Hachitani et al. (1981)(3) Inactive (3) Jensen et al. (1991)Inactive Roehm GMbH (1989a)Inactive Roehm GMbH (1989a)Inactive Roehm GMbH (1989b)Inactive Roehm GMbH (1989c)(1) Positive (1) MHW-Japan (1997)(2) Inactive (2) Dow Chemical (1995)Inactive Roehm GMbH (1989d)Inactive Roehm GMbH (1989e)

known Inactive EU (2001b)


cursor (epoxide) with a specific structural alert (Crameret al., 1978; Cheeseman et al., 1999) which is known to pro-duce a mutagenic response in this test.

In contrast to the bacterial reverse mutation and otherpoint mutation assays, virtually all of the acrylates/methac-rylates demonstrate a mutagenic response in the mouselymphoma assay. The mouse lymphoma test scheme hasthe capacity to provide information on both induction ofmutants arising from small DNA events (point mutations)as well as from possible larger mutations involving chro-mosomal damage (clastogenicity) as determined by the size(diameter) of mutant colonies formed. Increases seen pref-erentially in small mutant colonies are now broadly recog-nized as linked to a clastogenic mechanism associated withthe test material, rather than induction of point mutations(Moore et al., 1985). Mouse lymphoma cell cultures havealso been used independent of inclusion of the tk± locusto evaluate chromosomal effects.

Moore et al. (1988), testing acrylic acid and four mono-functional acrylates and methacrylates (methyl acrylate,ethyl acrylate, methyl methacrylate and ethyl methacrylate)in the mouse lymphoma assay, confirmed aberrationsinvolving chromatid- and chromosome-type rearrange-ments and chromosome-type breaks for each substanceas the increased mutant colony responses observed wererelated to increases in small colonies rather than large col-onies of mutant cells. Additional studies with several moreacrylate and methacrylate esters (2-hydroxyethyl acrylate,dicyclopentenyloxyethyl acrylate, tetraethyleneglycoldiacrylate, trimethylolpropane triacrylate, pentaerythritoltriacrylate, tetraethylene glycol dimethacrylate and tri-methylolpropane trimethacrylate) further confirmedincreases in small colony formation in the mouse lym-phoma assay (Dearfield et al., 1989). Based on mutationspectral analysis, Schweikl and Schmaltz (1999) reportedthe induction of large deletions in the hgprt gene of V79cells following exposure with the multifunctional acrylate,TEGDMA, and further reported identification of identicaltypes of chromosomal aberrations and the formation ofmicronuclei following in vitro testing of several acrylatesand methacrylates (Schweikl et al., 1998). Thus, theresponse seen with acrylates and methacrylates in vitro inthe mouse lymphoma assay appears related to effects onchromosomal numbers or structure rather than directinduction of point mutations.

Overall, a similar pattern of response has emerged forboth acrylates and methacrylates in the mouse lymphomaassay. Significant cytotoxicity is observed, generally witha nonlinear dose response both with and without activa-tion. Increases in mutations were observed at concentra-tions producing greater than 50% growth inhibition,while at least doubling of mutant frequency did not occurin cultures showing greater than 50% relative total growth.

The results of in vitro mouse lymphoma assays, raisingthe concern of mutagenicity, are clarified by results ofin vivo testing. The behavior of both monofunctional andmultifunctional acrylates and methacrylates in the mouse

micronucleus test and in the in vivo chromosomal aberra-tion assay indicate without equivocation that these chemi-cals are nongenotoxic in the whole animal. These in vivo

tests, measuring chromosomal effects rather than genemutation events, are directly appropriate to further eluci-date the nature of the presumed chromosomal effects assuggested by the increase in small, not large, colonies inthe mouse lymphoma assay. There is essentially no concor-dance between results of in vitro cytogenetics testing andresults observed in vivo for the same mutagenic endpoint.There are also consistent negative results in the in vivo

unscheduled DNA synthesis assay. Thus, consideration ofthe weight-of-evidence supports the conclusion that thesesubstances do not pose an in vivo mutagenic risk.

There are a number of conditions which may lead to apositive in vitro finding resulting in non-concordance withnegative (inactive) in vivo genotoxicity testing or whole ani-mal bioassay results. A number of these conditions havebeen delineated (ICH Harmonized Tripartite Guideline-S2A, 1995). Of those mentioned, there are several condi-tions of particular interest in review of acrylate and meth-acrylate group results in the mouse lymphoma assay vs.in vivo clastogenicity studies. These include: (a) possiblemetabolic conditions inherent in whole animals whichcan influence the in vivo study outcome, (b) possible non-DNA target interaction with a chemical that could indi-rectly result in genotoxicity in vitro, and (c) the influenceof high levels of cytoxicity leading to extreme culture con-ditions which do not occur in vivo.

Regarding metabolic conditions, both acrylates andmethacrylates (data generally available on the monofunc-tionals) are known to be rapidly absorbed, distributedand eliminated. In vivo experiments have demonstratedthat rats excrete between 50% and 70% of an acrylic esteras CO2 within 24 h following gavage administration (Gha-nayem et al., 1987; Bratt and Hathaway, 1977; Bereznow-ski, 1995). The primary route of detoxification involveshydrolysis by carboxylesterases found in a number of tis-sues which result in the release of acrylic or methacrylicacid and the corresponding alcohol. A similar metabolicpattern is observed in vitro. This reaction occurs rapidly,especially with short-chain esters, and is expected to occurquickly for multifunctional esters as well. In vivo, the corre-sponding acid is then metabolized to carbon dioxide bynormal oxidative catabolic pathways while the alcoholmoiety is metabolized via the endogenous alcohol dehydro-genase enzyme system. Thus, even though micronucleusassays with various acrylates and methacrylates have beenconducted at dose levels resulting in overt toxicity or evenup to the maximum guideline dose recommended for sucha study, significant detoxification mechanisms and rapidclearance in vivo may influence the duration and dose avail-able at the study target site.

Following testing of several acrylate and methacrylateesters in the mouse lymphoma assay, Dearfield et al.(1991) suggested that acrylates and methacrylates mayresult in alkylation of critical cellular nucleophiles, particu-


larly at doses where glutathione depletion was observed.However, Ciaccio et al. (1998), investigating mouse lym-phoma cells exposed to a range of test concentrations ofethyl acrylate, identified no single-strand DNA breaks inan alkaline elution assay while DNA double-strand breaksoccurred only in a pulsed-field gel electrophoresis test attest concentrations yielding significant cytotoxicity (>80%reduction in the RCG). Also, contrary to the Dearfieldet al. hypothesis, no toxic intermediates are evident in themitochondrial metabolism of acrylic acid and, importantly,the acid is not electrophilic in vivo under physiological con-ditions (McCarthy and Witz, 1997). Hence, direct DNAinteraction appears unlikely.

Involvement of non-DNA target tissue interactionremains a plausible explanation of the mouse lymphomatest results as conjugation of an acrylate or methacrylatemay occur spontaneously via a Michael addition of a vinylgroup to the sulfhydryl of glutathione (GSH), or by anenzymatic reaction catalyzed by GSH transferase. Reduc-tion of non-protein sulfhydryl (NPS) levels and bindingto GSH has been reported in vitro as well as in tissues ofexperimental animals exposed to acrylates. Coupled withthe effects of high cytotoxicity, in vitro depletion of a keyenzyme(s) could lead to cellular toxicity. Specifically usingmouse lymphoma cells, Ciaccio et al. (1998) observed thatthe ethyl acrylate-induced mutagenic response correlatedwith cellular cytotoxicity (apoptosis and necrosis) mediatedby NPS depletion and resulted in mitochondrial membraneimpairment, as measured by rhodamine uptake. It wouldthus appear that in vitro, significant NPS depletion and cel-lular disruption could lead indirectly to mutational eventsthat would not be expected in vivo at lower, more physio-logically relevant levels.

In the context of mutagenicity testing, the acrylatesbehave as a single category, even in the matter of the highlyconsistent positive results in the mouse lymphoma assay.Genotoxicity behavior of a similar compound withoutstructural alerts is thus predicted by its inclusion in thisclass. Acrylates and methacrylates are recognized as achemical category by the U.S. EPA in evaluation ofpremanufacturing notices, specifically in consideration ofenvironmental toxicity (EPA, 2006). Similarly, the OECDhas assessed short-chain alkyl methacrylate esters as achemical category within the HPV program. The EuropeanUnion has established a category of monoalkyl- and mono-aryl- acrylates, which it uses to regulate chemical classifica-tion and labeling within Annex 1 of the DangerousSubstances Directive (EEC, 1967). Acrylates and methac-rylates have been used as a legitimate category for the eval-uation of over 22 individual chemicals for GESAMP, aEuropean program of scientific experts concerned withmarine pollution (ECB, 2005). Data presented in this paperprovide additional validation of the concept of an ‘‘acry-late/methacrylate” category and further extends it toinclude the endpoint of mutagenicity.

Kirkland and colleagues (2005) undertook an evaluationrecently on the ability of a battery of three of the most

commonly used genotoxicity studies to discriminate rodentcarcinogens and non-carcinogens from among a large database of chronic rodent bioassays. The mouse lymphomaassay was found to have a high sensitivity (ability to iden-tify animal carcinogens) but an unacceptably low specific-ity (ability to demonstrate a negative mutagenic responsewith non-carcinogens). There was low concordance (highrates of false positives) between mouse lymphoma testresults in vitro and rodent carcinogenicity bioassay results.The authors concluded that ‘‘. . .an appropriate balancebetween sensitivity and specificity therefore needs to befound, as a basis for judging whether individual tests, orcombinations of tests, provide the best information fromwhich to make decisions”. On the basis of the lack of con-cordance of the mouse lymphoma assay, it was concludedthat some results may not be biologically relevant (Kirk-land et al., 2005). Similarly, based on low specificity seenafter evaluation of a series of short-term mutagenicity stud-ies previously used extensively by the U.S. NTP, both thesister-chromatid exchange (SCE) assay and the mouse lym-phoma assay have been removed from the present NTPrecommended short-term mutagenicity testing battery (Zei-ger, 2000). It is concluded that positive responses seen inthese assays may be considered of questionable biologicalrelevance.

We include 18 in vivo studies on the acrylate/methacry-late family, specifically addressing these cytogenetics end-points, with the conclusion that there is no concordancewith in vitro cytogenetics testing in mammalian cells.Kirkland et al. (2005, 2006) have concluded that ‘‘disap-pointing findings of this analysis [i.e., use of an in vitro

genotoxicity study battery to predict rodent cancer bioas-say outcome] suggests a complete rethink[ing] on in vitro

genotoxicity testing”. While sensitivity of the 3-test EUbattery (bacterial reverse mutation, mouse lymphomaand chromosomal aberration assay) was high, with over90% of the rodent carcinogens being detected by at leastone of the three tests, the specificity of this battery of testsresulted in a disturbing finding. While the bacterialreverse mutation assay specificity was reasonable (74%),all mammalian cell tests had very low (<45%) specificity,resulting in a very poor predictability of cancer bioassayoutcome. When multiple test results were considered, asfor sensitivity, the specificity further declined to unaccept-ably low levels. When all three EU-recommended testswere combined, 75–95% of all non-carcinogens gave amutagenic signal in at least one test included in the bat-tery. These results highlight deficiencies in use of the cur-rent battery of tests for prediction of carcinogenicresponse in whole animals (Kirkland et al., 2005).

Chronic rodent bioassay data on acrylates and methac-rylates provides a consistent theme of discordance betweennegative (inactive) bioassay results and positive responsesseen in some in vitro mutagenicity assays. It is recognizedthat a number of chemicals with the acryl moiety are con-sidered animal carcinogens, including acrylonitrile, acro-lein and methacrylonitrile; hence the structural alert


frequently applied to chemicals containing the acryl moi-ety. However, rodent bioassays with several acrylates/methacrylates have generated results showing a lack oftumorigenic response. Acrylic acid itself, when tested eitherorally in rats (Hellwig et al., 1993) or dermally in mice(DePass et al., 1984) was noncarcinogenic. Similarly, whentested via inhalation in rats, n-Butyl acrylate (Anon., 2005)and 2-hydroxyethyl acrylate (Rampy et al., 1978) resultedin no observable neoplastic effects. When tested by the der-mal exposure route in chronic rodent bioassays, no evi-dence of carcinogenicity was observed in studies withtriethyleneglycol diacrylate (TREGDA) (Van Milleret al., 2003) and n-Butyl acylate (DePass et al., 1984) whilestudies in two mice strains with 2-ethylhexyl acrylateresulted in conflicting results (Wenzel-Hartung et al.,1989; Mellert et al., 1994). Additionally, limited dermalcancer studies in mice with a series of monofunctionaland multifunctional acrylates resulted in no evidence ofsystemic tumor formation (Anderson and Clary, 1986).With regards to methacrylates, no treatment-relatedincreases in tumors were observed in rats, mice or hamstersexposed by inhalation for a lifetime to methyl methacrylate(NTP, 1986b; Lomax et al., 1997). TriethyleneglycolDimethacrylate (TREGDMA), tested in a mouse dermalbioassay (Van Miller et al., 2003) produced no skin tumorsand, in a limited oral study in rats, glycidyl methacrylatereportedly exhibited no evidence of carcinogenicity(BIBRA, 1988).

Special attention need be given to ethyl acrylate for, dueto numerous toxicity, metabolic and mechanistic datadeveloped on this chemical to better understand the impli-cations of its toxicological properties, it has become thesentinel chemical for understanding the toxicologicalprofile of acrylates and methacrylates as a whole. Chronicrodent studies have been conducted by the oral gavage,oral drinking water and inhalation exposure routes(Borzelleca et al., 1964; NTP, 1986a; Miller et al., 1985).When administered by gavage, ethyl acrylate producedtumors of the forestomach in mice and rats, although notat other systemic sites (NTP, 1986a). Administration torats in the drinking water resulted in no discernable tumor-igenic finding (Borzelleca et al., 1964). Additionally, whenadministered by inhalation, no carcinogenic response wasobserved in either rats or mice (Miller et al., 1985). Mech-anistic data developed with ethyl acrylate has establishedthat proliferation of the epithelial cell lining of the fore-stomach resulted in tumor formation (Ghanayem et al.,1990) and that this relationship between ethyl acrylate-induced forestomach tumor formation and cell prolifera-tion was temporally dependent (Ghanayem et al., 1994).Sustained increases in forestomach hyperplasia caused bygavage dosing resulted, so long as exposure continued.Upon cessation of exposure, regression of cellularproliferation occurred and development of forestomachtumors did not occur. These studies further confirmed theorgan specificity of the cell proliferation only at the tissuecontact site (forestomach) and not systemically in other

tissues. Extensive metabolic fate studies conducted withethyl acrylate both in vitro and in vivo have established itsmetabolic profile both at high and low dosages. High dos-age (i.e., gavage) administration is concordant with a toxictissue response only at the site of dosing resulting in severeglutathione depletion. Depletion of NPSH (non-proteinthiols) in the forestomach resulted in a series of cellulartoxicity events (edema, inflammation, ulceration, hyperpla-sia) leading to tumor development. Lower gavage dosescausing minimal changes in glutathione concentrationresulted in no local tissue toxicity and no tumors. Rapidsystemic detoxification even at high doses resulted in noevidence of toxicity in tissues remote from the site of dos-ing. Results of this work have been summarized in develop-ment of a physiologically-based model describing thechemical tissue interaction and its implications for riskassessment (Frederick et al., 1992).

Ethyl acrylate has previously (1989) been listed as a sub-stance ‘‘reasonably anticipated to be a human carcinogen”

in the Report on Carcinogens prepared by the NationalToxicology Program (NTP), a decision based on the resultsof the gavage studies in rats and mice. In 2000, ethyl acry-late was de-listed from the Report following extensive sci-entific review of the available chronic rodent, metabolismand mechanistic studies. It was concluded that the fore-stomach carcinogenicity observed in the gavage studiesrepresented the only treatment-related tumorigenicresponse in the various animal studies considered and thatthe irritation, hyperplasia and tumor responses in the fore-stomach were related to high target tissue concentrations ofethyl acrylate given by direct gavage addition rather thanto overall delivered dose. It was further considered thatstop-exposure studies, i.e., gavage doses of ethyl acrylatesufficient to induce sustained mucosal hyperplasia in theforestomach, had to be administered for longer than6 months before forestomach neoplasia would occur. Thistemporal and dose-dependent process for induction offorestomach tumors is considered the result of a nongeno-toxic mechanism of tumor formation (Butterworth, 1989).As such, these results provide further insight into the lackof in vivo mutagenic response seen with other acrylatesand methacrylates. Thus, looking at the predictability ofcarcinogenicity based on both the genotoxicity results,in vitro and in vivo, along with the carcinogenicity findingson structural analogues such as ethyl acrylate, can form auseful basis for a weight-of-evidence approach to assessthe broader scope of acrylate and methacrylate esters.

Kirkland et al. (2005) affirmed that an appropriate bal-ance between sensitivity and specificity needs to be foundfor any single or multiple test battery use for decision-mak-ing on biological relevance and prediction of carcinogenic-ity. Based on their review, the bacterial reverse mutationassay demonstrated the best specificity of all the in vitro

tests examined. This appears to be precisely the case forthe acrylate/methacrylate family. Based on our analysis,any additional in vitro mammalian cell testing of the acry-lates and methacrylates appears noninformative, and even


counterproductive, as the ability to predict the outcome ofin vivo tests is poor.

When category-based information predicts the result ofan in vitro mammalian assay to be positive, preference maybe given to supplementary tests, such as the second tierin vivo assay. If the in vitro mutagenicity testing is designedprimarily for hazard identification purposes, this hazardcan clearly be appreciated from the category data.

Use of secondary tier study results to reconcile primarytier discrepancies appears to have been adopted by the EUScientific Committee on Food (SCF), when it concludedthat acrylic acid was not genotoxic in vivo and thus posedneither a mutagenic nor carcinogenic risk. This was basedon data available on acrylic acid itself, and also on a con-clusion of non-mutagenicity seen in in vivo bone marrowcytogenetics assays with other monofunctional acrylatesand methacrylates (methyl methacrylate, methyl acrylate,ethyl acrylate and butyl acrylate) within the category(EU, 2001a).

EFSA has listed acrylic acid and 11 additional mono-functional derivatives as approved as food contact materi-als, even in the presence of active mouse lymphomafindings (European Commission, 2002), with a total spe-cific migration limit of 6 mg/kg. The same total specificmigration limit of 6 mg/kg has been determined for meth-acrylic acid and 12 monofunctional methacrylates. It ishighly likely that the consistently inactive response seenin the in vivo studies provided the pivotal evidence neededfor EFSA to conclude that these monofunctional acrylatesand methacrylates possessed no appreciable mutagenic riskto human health. It would appear that sufficient data existson structurally similar esters to use ‘‘read across” to con-sider other monofunctional acrylates and methacrylates.EFSA recently added 2-ethylhexyl acrylate (EHA) to a listof approved food contact materials, adopting the ‘‘grouprestriction” limits (EFSA Journal, 2004). Trimethylolpro-pane trimethacrylate, a multifunctional methacrylate, wasgiven EU approval for food contact use based on reviewand conclusions reached at the 130th SCF meeting (EU,2001b). The Committee recognized the results obtainedfrom in vitro bacterial and mammalian cell gene mutationassays cited above, as well as lack of mutagenic responseseen in an in vivo micronucleus assay and an in vivo/

in vitro UDS assay in rat liver.It should be realized that most of the in vivo data avail-

able has been conducted with monofunctional acrylatesand methacrylates rather than their multifunctional coun-terparts. For these latter subgroups, the supporting dataare modest as even those studies completed with multifunc-tional acrylates have been by the dermal route (with oneexception—Trimethylolpropane triacrylate), where assur-ance of adequacy of test material at the target site is notfully substantiated. It is likely that results of several addi-tional studies, by the oral route to establish target siteexposure, would allow regulatory agencies to accept as suf-ficient the testing for this endpoint amongst the largergroup of multifunctional acrylates and methacrylates.

5. Conclusions

An extensive genotoxicity testing (in vitro and in vivo

studies) database has been developed and reported on alarge number of structurally similar mono- and multifunc-tional esters of acrylic acid and methacrylic acid. With fewexceptions, these compounds demonstrate consistentlynonmutagenic results in the bacterial reverse mutationassay and other in vitro mammalian point mutation assays.They also show a pattern of positive response in in vitro

mammalian clastogenicity assays, but are consistently inac-tive in in vivo assays (micronucleus) designed to assess clas-togenic potential in whole animals. Mechanistic studieshave confirmed that the organ-specific tumors induced byethyl acrylate are the result of a non-genotoxic mechanism,while non-tumorigenic findings from a series of chronicrodent bioassays conducted with acrylic acid and severalacrylate and methacrylate esters provides further supportfor their lack of genotoxic potential in the whole animal.Thus, a critical analysis of experimental conditions, com-bined with a weight-of-the-evidence approach, clarifiesthe status of the acrylates and methacrylates as nonmuta-genic in whole animals.

Acrylates and methacrylates behave as a chemical familywhen studied for genotoxicity potential and, with fewexceptions based on alerting chemical structures, new com-pounds in this family may be considered for waiver fromactual testing based on structure–activity relationships.

These data support and further extend the work of Kirk-land et al., 2005, in their call for a general reevaluation ofthe utility of a battery of in vitro mammalian mutagenicityand clastogenicity testing in safety evaluation, particularlyas it applies to the esters of acrylic or methacrylic acid.

Acknowledgment

The authors acknowledge the technical input and criticalreading of the manuscript by Francoise Godts, Ph.D. andWendy Wellens, M.S.

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