29
Fundamental and Molecular Mechanisms of Mutagenesis ELSEVIER Mutation Research 330 (1995) 71-99 Acrylamide: a review of its genotoxicity and an assessment of heritable genetic risk * Kerry L. Dearfield a,*, George R. Douglas b, Udo H. Ehling ‘, Martha M. Moore d, Gary A. Sega e, David J. Brusick f a U.S. Em~ironmental Protection Agency, Office of Pesticide Programs (7509C), 401 M Street, S. W., Washington, DC 20460, USA b Health Canada, Environmental Health Centre, Ottawa, Canada ’ GSF-Institut fiir Siiugetiergenetik, Forschungszentrum fir Umwelt und Gesundheit, Neuherberg, Germany d U.S. Emlironmental Protection Agency, Health Effects Research Laboratory, Research Triangle Park, NC, USA e Oak Ridge National Laboratory, Analytical Chemistry Division, Oak Ridge, TN, USA f Hazleton Washington. Inc., Vienna, VA, USA Accepted 6 February 1995 Abstract An updated review of the genotoxicity studies with acrylamide is provided. Then, using data from the studies generating quantitative information concerning heritability of genetic effects, an assessment of the heritable genetic risk presented by acrylamide is presented. The review offers a discussion of the reactions and possible mechanisms of genotoxic action by acrylamide and its epoxide metabolite glycidamide. Several genetic risk approaches are discussed, including the parallelogram, direct (actually a modified direct), and doubling dose approaches. Using data from the specific-locus and heritable translocation assays, the modified direct and doubling dose approaches are utilized to quantitate genetic risk. Exposures of male parents to acrylamide via inhalation, ingestion, and dermal routes are also quantitated. With these approaches and measurements and their underlying assumptions concerning extrapolation factors (including germ cell stage specificity, DNA repair variability, locus specificity), number of human loci associated with dominant disease alleles, and spontaneous mutation rates, an assessment of heritable genetic risk for humans is calculated for the three exposure scenarios. The calculated estimates for offspring from fathers exposed to acrylamide via drinking water are up to three offspring potentially affected with induced genetic disease per lo8 offspring. Estimates for inhalation or dermal exposures suggest higher risks for induced genetic disease in offspring from fathers exposed in occupational settings. Keywords: Acrylamide; Genetic risk *This manuscript has been reviewed by the Office of Prevention, Pesticides and Toxic Substances (U.S. Environ- mental Protection Agency), the Health Effects Research Lab- oratory (U.S. Environmental Protection Agency), and the Bureau of Chemical Hazards (Health Canada) and approved for publication. Approval does not signify that the contents necessarily reflect the views or policies of these agencies, nor does mention of trade names or commercial products consti- tute endorsement or recommendation for use. * Corresponding author. Tel. 703 305 6780; Fax 703 305 5453. 0027-5107/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0027-5107(95)00037-2

Acrylamide: a review of its genotoxicity and an assessment of heritable genetic risk

Embed Size (px)

Citation preview

Fundamental and Molecular Mechanisms of Mutagenesis

ELSEVIER Mutation Research 330 (1995) 71-99

Acrylamide: a review of its genotoxicity and an assessment of heritable genetic risk *

Kerry L. Dearfield a,*, George R. Douglas b, Udo H. Ehling ‘, Martha M. Moore d, Gary A. Sega e, David J. Brusick f

a U.S. Em~ironmental Protection Agency, Office of Pesticide Programs (7509C), 401 M Street, S. W., Washington, DC 20460, USA b Health Canada, Environmental Health Centre, Ottawa, Canada

’ GSF-Institut fiir Siiugetiergenetik, Forschungszentrum fir Umwelt und Gesundheit, Neuherberg, Germany d U.S. Emlironmental Protection Agency, Health Effects Research Laboratory, Research Triangle Park, NC, USA

e Oak Ridge National Laboratory, Analytical Chemistry Division, Oak Ridge, TN, USA f Hazleton Washington. Inc., Vienna, VA, USA

Accepted 6 February 1995

Abstract

An updated review of the genotoxicity studies with acrylamide is provided. Then, using data from the studies generating quantitative information concerning heritability of genetic effects, an assessment of the heritable genetic risk presented by acrylamide is presented. The review offers a discussion of the reactions and possible mechanisms of genotoxic action by acrylamide and its epoxide metabolite glycidamide. Several genetic risk approaches are discussed, including the parallelogram, direct (actually a modified direct), and doubling dose approaches. Using data from the specific-locus and heritable translocation assays, the modified direct and doubling dose approaches are utilized to quantitate genetic risk. Exposures of male parents to acrylamide via inhalation, ingestion, and dermal routes are also quantitated. With these approaches and measurements and their underlying assumptions concerning extrapolation factors (including germ cell stage specificity, DNA repair variability, locus specificity), number of human loci associated with dominant disease alleles, and spontaneous mutation rates, an assessment of heritable genetic risk for humans is calculated for the three exposure scenarios. The calculated estimates for offspring from fathers exposed to acrylamide via drinking water are up to three offspring potentially affected with induced genetic disease per lo8 offspring. Estimates for inhalation or dermal exposures suggest higher risks for induced genetic disease in offspring from fathers exposed in occupational settings.

Keywords: Acrylamide; Genetic risk

*This manuscript has been reviewed by the Office of

Prevention, Pesticides and Toxic Substances (U.S. Environ-

mental Protection Agency), the Health Effects Research Lab-

oratory (U.S. Environmental Protection Agency), and the

Bureau of Chemical Hazards (Health Canada) and approved

for publication. Approval does not signify that the contents

necessarily reflect the views or policies of these agencies, nor

does mention of trade names or commercial products consti-

tute endorsement or recommendation for use.

* Corresponding author. Tel. 703 305 6780; Fax 703 305

5453.

0027-5107/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved

SSDI 0027-5107(95)00037-2

72 K.L. Dearfield et al. /Mutation Research 330 (1995) 71-99

1. Introduction

This acrylamide assessment is the culmination of an effort emanating from an October, 1993 joint European Commission (EC)/United States Environmental Protection Agency (USEPA) Workshop on Risk Assessment examining ‘Hu- man Genetic Risks from Exposure to Chemicals, Focusing on the Feasibility of a Parallelogram Approach’ (USEPA, 1994; for meeting report, Waters and Nolan, 1994). Workgroups were formed to review the genetic toxicity data for acrylamide, ethylene oxide, 1,3-butadiene, and cy- clophosphamide. Using the available data and exposure information, the feasibility of conduct- ing genetic risk assessments for adverse heritable effects was examined for the four compounds. The work of the acrylamide group (see author list) is contained in this report.

Since the earlier review of the genetic toxicity of acrylamide was published (Dearfield et al., 19881, a great number of studies examining the somatic and germ cell risk from acrylamide expo- sure have been published. Many of these studies provide data that allow one to explore ap- proaches for assessing heritable risks from expo- sure to monomeric acrylamide. In addition, expo- sure assessments for acrylamide have been con- ducted by other groups and provide human expo- sure estimates for use in calculating genetic risk estimates.

Acrylamide is an important industrial com- pound with many uses; for instance, polyacryl- amides are used as flocculents for wastewater treatment, in adhesives and grouts, and in labora- tory gels (see Dearfield et al., 1988 and new International Agency for Research on Cancer (IARC) monograph on acrylamide, in press, for more detail). Thus, there are many opportunities for individuals to be exposed to monomeric acryl- amide either in its production or in its use. Be- cause of the known human exposure and the genetic effects induced by acrylamide in animals, it is important to examine its potential for induc- ing adverse heritable effects in exposed human beings.

The present analysis builds upon preceding group efforts as well as the individual efforts

referenced in this report. The International Com- mission for Protection Against Environmental Mutagens and Carcinogens (ICPEMC) has pro- duced two reports: one for the Department of Health, Canada (ICPEMC, 1993a) and the other for the USEPA (ICPEMC, 1993b). These reports develop genetic risk extrapolation models, and the former report uses acrylamide data in pre- senting practical examples of risk estimation cal- culations. A detailed description of the ICPEMC genetic risk extrapolation models is published elsewhere in this issue (Favor et al.). The present Workgroup expanded upon the ICPEMC risk cal- culation for acrylamide using different exposure scenarios for its genetic risk assessment.

2. Update on genotoxicily studies

A large number of studies have been reported since the last published review of the acrylamide genetic toxicity data (Dearfield et al., 1988). While the ICPEMC group recently reviewed the litera- ture (ICPEMC, 1993a), that report remains un- published. Table 1 provides a compilation of the new studies since the last published review (the table includes several studies not found in the ICPEMC review, but does not include the studies presented in the previously published review). Table 2 presents the studies dealing with the genetic toxicity of glycidamide, the epoxide metabolite of acrylamide (discussed in section 3). As this report deals primarily with the use of the studies to assess potential heritable risk to hu- mans, the genetic toxicity of acrylamide and glyci- damide is discussed briefly below.

Gene mutation assays Acrylamide has consistently exhibited negative

results in bacterial gene mutation assays with and without activation. The available literature in- cludes the use of many strains of Salmonella (Zeiger et al., 1987; Tsuda et al., 19931, Es- cherichia coli (Tsuda et al., 1993), and Klebsiella pneumoniae (Knaap et al., 1988) and report test- ing at extremely high concentrations of acryl- amide (up to 100 mg/plate). On the other hand, glycidamide induced a positive response in

K. L. Dearfield et al. /Mutation Research 330 (1995) 71-99 73

Salmonella strains TAlOO and TA1535 (Hashimo- to and Tanii, 1985), but not in K. pneumoniae (Voogd et al., 1981).

In in vitro mammalian gene mutation assays, acrylamide showed activity at the tk locus in mouse lymphoma cells (Barfknecht et al., 1988; Knaap et al., 1988). There were some positive

,results with mouse lymphoma cells at the hprt locus (Knaap et al., 19881, but negative results were reported at the hprt locus in V79 cells (Tsuda et al., 1993). As suggested in one of the earlier reports using the L5178Y/tk mouse lym- phoma assay (Moore et al., 1987), the activity may be primarily due to clastogenic effects (almost all induced tk mutants were small colonies and gross aberrations were induced); however, point muta- tions may also be induced (see below). Glyci- damide also induced mutations at the tk locus of mouse lymphoma cells without exogenous activa- tion (Barfknecht et al., 1988).

spermatids), but not in spermatogonia. Cytoge- netic evidence demonstrated that most of the specific-locus mutations were multi-locus (large) lesions. In the Ehling and Neuhauser-Klaus (1992) report, mice were exposed to a single dose of 100 mg or 125 mg acrylamide/kg. Not only were post-spermatogonia stages affected, but specific- locus mutations were also detected in the sper- matogonia at 100 mg/kg. The spermatogonia ef- fects at 100 mg/kg will be used in the calcula- tions in section 5; the data are:

Exposure Number of Number of regime mutations offspring

Historical control 22 248413 100 mg/kg 6 23 489

Structural chromosomal alterations

Both somatic mutations and sex-linked reces- sive mutations have been observed in Drosophila melanogaster after larval feeding of acrylamide (Knaap et al., 1988; Batiste-Alentorn et al., 1991; Tripathy et al., 1991). This suggests a point muta- tion mechanism for acrylamide in an in vivo situa- tion. However, no sex-linked recessive mutations were detected in D. melanogaster after a single abdominal injection (Knaap et al., 1988).

Several studies report the induction of chro- mosomal aberrations and mitotic disruptions fol- lowing acrylamide exposure of cultured mam- malian cells, mostly Chinese hamster cell strains and lines (Sofuni et al., 1985; Knaap et al., 1988; Warr et al., 1990; Tsuda et al., 1993; Llhdetie et al., 1994). Reported polyploidy and spindle dis- turbances suggest that acrylamide could have ef- fects related to aneuploidy (Warr et al., 1990; Adler et al., 1993; Tsuda et al., 1993).

In vivo experiments with several strains of mice In the early studies of acrylamide, it was sug- suggest the potential of acrylamide to induce gested that acrylamide was a specific germ cell gene mutations. A weak (almost equivocal) re- mutagen (Shiraishi, 1978). As a result, there has sponse was found for 1acZ mutations in a been a great deal of experimentation with in vivo Muta@Mouse experiment (Hoorn et al., 1993). A somatic and germ cells following acrylamide ex- mouse spot test was positive with the induction of posure. It has subsequently been demonstrated colored spots (though not only point mutations, that induction of aberrations and micronuclei in but chromosomal mutations, whole chromosome mouse bone marrow occurs (Adler et al., 1988; loss, or somatic recombination could account for Cihak and Vontorkov& 1988, 1990; Knaap et al., induced spots) (Neuhauser-Klaus and Schmahl, 1988) as well as micronuclei in peripheral blood 1989). Acrylamide produced positive responses in reticulocytes (Russo et al., 1994). In addition, the morphological specific-locus test, a test that micronuclei (but not aberrations) in spleen lym- can be used to provide quantitative data in a phocytes (Backer et al., 1989) and splenocytes heritable risk assessment for humans (Russell et (Kligerman et al., 1991) have been observed. Be- al., 1991; Ehling and Neuhauser-Klaus, 1992). In cause no aberrations were seen in the spleen the Russell et al. (1991) report, mice were ex- studies, the micronucleus assays may be detecting posed for 5 days .at 50 mg acrylamide/kg/day. the loss of chromosome(s). This may account for Mutations were increased in the post-meiotic the differences between the aberration and mi- stages of spermatogenesis (spermatozoa and late cronucleus results in spleen. It is interesting to

74

Table 1 Acrylamide genotoxicity assays

Assay Test system

K L. Dearfield et al. /Mutation Research 330 (1995) 71-99

Concentration/ HID or LED b Assay result Dose a

Reference

(I) GENE MUTATION ASSAYS (IA) Bacterial gene mutation assays Salmonella S. typhimurium TA1535, 10-10000 pg/plate reversion TA1537, TA98, TAlOO assay (NTP first assay)

S. typhimurium TA1535, 100-10000 pg/plate TA97, TA98, TAlOO (NTP second assay)

S. typhimurium TAl535, l-100 mg/plate TA1537, TA98, TAlOO, TA102

S. typhimurium TA1535, 0.5-50 mg/plate TA1537, TA98, TAlOO

E. coli gene E. coli WP2 uvrA- 0.5-50 mg/plate mutation assay

Fluctuation test K. pneumoniae 2-10 mg/ml ur- pro-

(IB) In vitro mammalian gene mutation assays Mammalian Mouse lymphoma gene L5178Y TK+‘-, mutation tk locus assay

Mouse lymphoma L5178Y TK+‘-, tk locus

Mouse lymphoma L5178Y TK+‘-, hprt locus

Mouse lymphoma L5178Y TKf’-, hprt locus

Chinese hamster cell line (V791, hprt locus

(1C) In vivo gene mutation assays Sex-linked D. melanogaster

recessive Berlin K lethal assay D. melanogaster

Somatic muta- D. melanogaster tion and/or recombination assay

10 mM 10 mM

0.5-7.5 mg/ml

0.5-7.5 mg/ml

0.1-0.5 mg/ml; with co-cultivated mammalian cells

l-7 mM; no activation used

40-50 mM; abdominal injection

0.25-5 mM; larvae feeding

l-l.5 (concentration unit not stated); larvae feeding

100 pg/plate

10000 Kg/plate

100 mg/plate

50 mg/plate

50 mg/plate

10 mg/ml

7.5 mg/ml

2.0 mg/ml

0.3 mg/ml

7 mM (500

pg/mB

50 mM

1.0 mM

1.0

Weak positive (TA98, TAlOO with activation only); rest negative

Negative

Negative

Negative

Negative

Negative

Positive (more response without activation)

Equivocal with activation (increase only at low survival)

Equivocal (increase only at low survival)

Positive

Negative

Negative

Positive

Weak positive

Zeiger et al., 1987

Zeiger et al., 1987

Knaap et al., 1988

Tsuda et al., 1993

Tsuda et al., 1993

Knaap et al., 1988

Barfknecht et al., 1988

Knaap et al., 1988

Knaap et al., 1988

Knaap et al., 1988

Tsuda et al.. 1993

Knaap et al., 1988

Tripathy et al., 1991

Knaap et al., 1988

Table 1 (continued)

Assay Test system

KL. Dearfield et al. /Mutation Research 330 (1995) 71-99

Concentration/ HID or LED b Assay result Dose a

Reference

15

(1 C) In vivo gene mutation assays D. melanogaster UZ stock D. melanogaster

Transgenic Muta@Mouse mouse IacZ gene mutations

Mouse spot (T x HT)F, test mouse embryos;

T-stock parental pregnant females exposed

l-l.5 mM, larvae feeding 0.25-5 mM; larvae feeding

5 x 50 mg/kg/day; i.p. injection

1 x 50 or 75 mg/kg; 3 x 50 or 75 mg/kg/ day; i.p. injection

Morphological specific-locus test

Male (BH/ Rl x lOl/Rl)F, mice

Male (102/ El x C3H/ El)F, mice

5 x 50 mg/kg/day; i.p. injection

loo-125 mg/kg; i.p. injection

(2) STRUCTURAL CHROMOSOMAL ALTERATIONS

(2.4) In vitro chromosomal alterations in mammalian cells In vitro chromosomal aberrations

Mitotic cell division aberration assay

Micronucleus assay

Polyploidy

Chromosome enumeration assay

Chinese hamster cells (CHL)

Chinese hamster cell line (V79)

Chinese hamster cell line (V79)

Chinese hamster lung cell line DON:Wg3h

Low passage Chinese hamster lung fibroblast LUC2

Seminiferous tubular seg- ments (sperma- tids from male Sprague-Dawley rats)

Chinese hamster cell line (V79)

Low passage Chinese hamster lung fibroblast LUC2

0.1-l mg/ml

0.1-3 mg/ml

0.5-5 mM; no activation used

200-1000 pg/ml

10-509 pg/ml

5-50 pg/ml; no activation used

0.5-5 mM; no activation used

12.5-500 pg/ml

1.0 mM

1.0 mM

5 x 50

mg/kg/day

50 mg/kg; 3 x 50 mg/kg/day

50 mg/k/day

100 w/kg

0.15 mg/ml (-S9) 0.5 mg/ml ( + S9)

1 mg/ml c-s91 0.1 mg/ml (+ S9)

2mM

200 pg/ml

10 i.Wml

50 pg/ml

1mM Positive

500 pg/ml Positive

Positive

Positive

Weak positive

Positive

Positive (post- spermatogonia)

Positive (post- spermatogonia; spermatogonia)

Positive

Positive

Positive

Positive

Positive

Negative

Batiste-Alentorn et al., 1991 Tripathy et al., 1991

Hoorn et al., 1993

Neuhauser-Klaus and Schmahl, 1989

Russell et al., 1991

Ehling and Neuhluser- Klaus, 1992

Sofuni et al., 1985

Knaap et al., 1988

Tsuda et al., 1993

Warr et al., 1990

Warr et al., 1990

Lahdetie et al., 1994

Tsuda et al., 1993

Warr et al., 1990

76 RL. Deatfield et al. /Mutation Research 330 (1995) 71-99

Table 1 (continued)

Assay Test system Concentration/ HID or LED b Assay result Reference Dose a

0.01-l mg/ml 0.01 mg/ml (24) In vitro chromosomal alterations in mammalian cells

Spindle Chinese hamster disturbances cell line W79)

(2B) In vivo chromosomal alterations

Aberrations (bone marrow)

Aberrations (spleen lymphocytes)

Aberrations (splenocytes)

Aberrations (spermatogonia)

Aberrations (spermatocytes)

Aberrations (first cleavage embryos)

Aberrations (first cleavage one-cell zygotes)

Micronucleus assay (bone marrow)

Male and female (lOl/El x C3H/ El)F, mice

Male ICR-SPF mice

Male C57BL/ 65 mice

Male C57BL/ 6 mice

Male (lOl/El X C3H/El)F, mice

Male C57BL/ 65 mice

Male (102/El x C3H/ El)F, mice

Male (102/El x C3H/ El)F, mice

Male CF, mice exposed; after mating, embryos examined

Male B6C3Fl mice

SO-150 mg/kg; i.p. injection

50 mg/kg

100 mg/kg; i.p. injection

SO-125 mg/kg; i.p. injection

100 mg/kg

125 mg/kg

loo mg/k; 100 mg/kg i.p. injection

50-150 mg/kg; 150 mg/kg i.p. injection

50-125 mg/kg; 125 mg/kg i.p. injection

IO&125 mg/kg; 125 mg/kg; 5 X 50 mg/kg/day; 5 X 50 mg/kg/ i.p. injection day 100 mg/kg; 100 mg/kg i.p. injection

150 mg/kg; 150 mg/kg i.p. injection; embryos assayed after spermiogenic stages exposed

75 and 125 mg/kg; 75 mg/kg exposed; after mating, 5 X 50 mg/kg/day; zygotes examined i.p. injection

Male and female 50-125 mg/kg; 50 mg/kg (lOl/El x C3H/ i.p. injection El)F, mice

Male ICR-SPF 1 x 100 mg/kg; 100 mg/kg; mice 2 x 25-100 mg/kg/ 2 X 25 mg/kg/

day; i.p. injection day

Male and female 136 mg/kg; 136 mg/kg Swiss-NIH mice i.p. injection

Male and female 1, 2 or 3 X 42.5 or 42.5 mg/kg/ ICR-SPF mice 68 mg/kg/day (9 ); day (9 ); 55 mg/

1,2 or 3 X 55 or kg/day (6) 88 mg/kg/day (6); 1 or 2 x 100 mg/kg/ day (both sexes); i.p. injection

Micronucleus assay Male BALB/c (peripheral blood mice reticulocytes)

Micronucleus assay Male C57BL/ (spleen lymphocytes) 65 mice

50 and 100 mg/kg; 50 mg/kg i.p. injection

50-125 mg/kg; 50 mg/kg i.p. injection

Positive Adler et al., 1993

Positive Adler et al., 1988

Positive

Negative

Cihak and Vontorkova, 1988 Backer et al., 1989

Negative Kligerman et al., 1991

Negative

Negative

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Adler et al., 1988

Backer et al., 1989

Adler, 1990

Adler, 1990

Valdivia et al., 1989

Pacchierotti et al., 1994

Adler et al., 1988

Cihik and Vontorkova, 1988

Knaap et al., 1988

Cihak and Vontorkova, 1990

Russo et al., 1994

Backer et al., 1989

K L. Dea@eld et al. /Mutation Research 330 (1995) 71-99 77

Table 1 (continued)

Assay Test system Concentration/ Dose a

HID or LED b Assay result Reference

(28) In vivo chromosomal alterations

Micronucleus assay Male C57BL/ (splenocytes) 6 mice

Micronucleus assay Male C57BL/ (spermatid) 65 mice

Male Lewis rats

Male Sprague- Dawley rats

Male BALB/c mice

Synaptonemal complex aberrations (germ cells)

Synaptonemal complex irregularities (germ cells)

Dominant lethal assay

Male C57BL/6J mice

Male C57BL/6J mice

Male (C3H X 101) F, mice

Male Fischer 344 rat

Male Pzh: SFISS mice Male (102/El x C3H/ ElJF, mice Male CD-1 mice

Male (C3H/Rl x lOl/ Rl)F, mice

Heritable Male translocations (C3H x lOl)F,

mice

Male C3H/El mice

Reciprocal Male C3H/ translocations El mice

(3) OTHER GENOTOXIC EFFECTS

100 mg/kg; i.p. injection

10-100 mg/kg; i.p. injection

50 and 100 mg/kg; 4 x 50 mg/kg/day; i.p.injection

50 and 100 mg/kg; 4 x 50 mg/kg/day; i.p. injection

50 and 100 mg/kg; 4 x 50 mg/kg/day; i.p. injection

50-150 mg/kg; i.p. injection

50-150 mg/kg; i.p. injection

5 x 40 mg/kg/day; i.p. injection

5 x 30 mg/kg/day; gavage

75-125 mg/kg; 1.p. injection 50-125 mg/kg; 1.p. injection

0.72-9.2 mg/kg/day; drinking water for 20 weeks

5 X 25-125 mg/kg/ day; dermal

5 X 40-50 mg/kg/ day; i.p. injection

50 and 100 mg/kg; i.p. injection

5 X 50 mg/kg/day; i.p. injection

100 mg/kg

50 mg/kg

100 mg/kg

4 X 50 mg/kg/ day

Positive

50 mg/kg Positive

150 mg/kg

50 mg/kg

40 mg/kg/day

30 mg/kg/day

125 mg/kg

75 mg/kg

9.2 mg/kg/day

25 w/k/day

40 mg/kg/day

50 mg/kg

50 mg/kg/day

(3A) DNA damage and /or repair assays and related tests B. subtilis B. subtilis l-50 mg/disk ret assay spores

DNA breakage Male 25-125 mg/kg; (alkaline elution) (C3H x C57BLj i.p. injection

1OJFr mice

10 mg/disk

25 mg/kg

Positive

Positive

Positive

Negative

Weak positive (asynapsis in meiotic prophase)

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Kligerman et al., 1991

Collins et al., 1992

Xiao and Tates, 1994

LIhdetie et al., 1994

Russo et al., 1994

Backer et al.. 1989

Backer et al.. 1989

Shelby et al., 1987

Working et al., 1987

Dobrzytiska et al., 1990 Ehling and Neuhiuser-Klaus, 1992 Fail et al., 1992

Gutierrez-Espeleta et al., 1992

Shelby et al., 1987

Adler et al., 1994

Adler, 1990

Tsuda et al., 1993

Sega and Generoso, 1990

78

Table 1 (continued)

LL. DearfXd et al. /Mutation Research 330 (1995) 71-99

Assay Test system Concentration/ Dose a

(3A) DNA damage and /or repair assays and related tests

HID or LED b Assay result Reference

In vitro UDS

In viva/ in vitro UDS

In vivo UDS

DNA adducts

Primary rat hepatocytes Primary hepato- cytes from male Fischer 344 rats

Human mammary epithelial cell (HMEC)

Male Fischer 344 rat hepatocytes

Male Fischer 344 rat spermatocytes

Male (C3H X 1011 F, and (C3H X BLlOlF, mice germ cells

Male (C3H x BLlOlF, mice testis

Male (C3H x BLlO)F, mice liver

5-20 mM

0.01-l mM

17.5 mM

1mM

l-10 mM 1mM

1 x 100 mg/kg; 5 x 30 mg/kg/day; gavage

1 x 100 mg/kg; 5 x 30 mg/kg/day; gavage

7.8-125 mg/kg; i.p. injection

1 X 100 mg/kg; 5 x 30 mg/kg/

day

5 x 30 mg/kg/

day

7.8 mg/kg

46 me/kg; i.p. injection

46 mg/kg

46 mg/kg; i.p. injection

46 mg/kg

(3B) Sister chromatid exchange (SCE) assays In vitro SCE Chinese hamster cell 0.1-l mg/ml assays line (V79)

Chinese hamster cell line (V79)

In vivo SCE Male BALB/c assay mice (differentiating spermatogonial

In vivo SCE Male C57BL/6J assay (spleen mice lymphocytes)

In vivo SCE Male C57BL/ assay 6 mice (splenocytesl

0.5-2.5 mM; no activation used

50 and 100 mg/kg; i.p. injection

50-125 mg/kg; i.p. injection

100 m/kg; i.p. injection

(3C) Cell transformation Cell transfor- C3H/lOT; mation cells

BALB/c 3T3 cells

lo-300 pg/ml; no activation used? OS-2 mM, no activation used

0.3 mg/ml (without act.1 1.0 mg/ml (with act.)

1mM

50 mg/kg

50 mg/kg

100 mg/kg

300 pg/ml

1mM

Weak positive

Negative

Positive

Negative

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Positive

Negative

Barfknecht et al., 1988 Butterworth et al., 1992

Butterworth et al., 1992

Butterworth et al., 1992

Butterworth et al., 1992

Sega et al., 1990

Sega et al., 1990

Sega et al., 1990

Knaap et al., 1988

Tsuda et al., 1993

Russo et al.. 1994

Backer et al.. 1989

Kligerman et al., 1991

Abernethy and Boreiko, 1987 Tsuda et al., 1993

K L. Dearfeld et al. /Mutation Research 330 (1995) 71-W 79

Table 1 (continued)

Assay Test system Concentration/ Dose a

HID or LED b Assay result Reference

(30) Germ cell effects Sperm head Male DNA alky- (C3H x LOl)F, lation mice

Sperm head Male protamine (C3H x lOl)F, alkylation mice

Sperm head Male ddY mice abnormalities

125 mg/kg; i.p. injection

125 mg/kg; i.p. injection

0.3-1.2 mM; in drinking water for 4 weeks

125 mg/kg

125 mg/kg

1.2 mM

Weak positive

Positive

Positive

Sega et al., 1989

Sega et al., 1989

Sakamoto and Hashimoto, 1986

a In vitro assays performed with and without exogenous activation unless indicated otherwise or the test system does not normally use such supplementation; route is indicated for in vivo studies. b HID, highest ineffective concentration/dose for negative studies; LED, lowest effective concentration/dose for positive studies.

Table 2 Glycidamide genotoxicity assays

Assay Test system

(1) GENE MUTATIONASSAYS (IA) Bacterial gene mutation assays Salmonella S. typhimutium reversion TA1535, TA1537, assay TA98, TAlOO

Fluctuation test K. pneumoniae

Concentration/Dose a HID or LED b Assay result Reference

5-5000 pg/plate; plate incorporation and preincubation

50 mmol/l; no exogenous activation used

5000 pg/plate

50 mmol/I

Positive (TABOO Hashimoto and and TA1535 + S9) Tanii, 1985

Negative Voogd et al., 1981

(1B) In vitro mammalian gene mutation assays Mammalian gene Mouse lymphoma 2.5 mM mutation assay L5178Y TK+‘-,

tk locus

2.5 mM Positive without activation

(2) OTHER GENOTOXIC EFFECTS (2A) DNA damage and /or repair assays and related tests Ili vitro UDS Primary rat hepato- 4.0 mM

cytes Primary hepatocytes 0.1-10 mM from male Fischer 344 rats

4.0 mM

1.0 mM

Negative

Positive

Human mammary l-10 mM 1.0 mM epithelial cell (HMEC)

Positive

Bartknecht et al., 1988

Barfknecht et al., 1988

Buttenvorth et al., 1992

Butterworth et al., 1992

a In vitro assays performed with and without exogenous activation unless indicated otherwise or the test system does not normally use such supplementation; route is indicated for in vivo studies. b HID, highest ineffective concentration/dose for negative studies; LED, lowest effective concentration/dose for positive studies.

80 KL. Dearfield et al. /Mutation Research 330 (1995) 71-99

note that while there is generally a dose response for the bone marrow data, the response is not very large compared to the responses seen in germ cells. This suggests that while acrylamide may not be a specific germ cell mutagen, it may preferentially affect germ cells versus somatic cells.

Acrylamide has proven to be genotoxic to germ cells. While aberration studies with spermatogo- nia have not detected an induced response (Adler et al., 1988; Backer et al., 1989; Adler, 19901, most structural chromosomal alteration studies evaluating other stages of spermatogenesis are positive. Aberrations were detected in spermato- cytes (Adler, 1990) and micronuclei were de- tected in spermatids (Collins et al., 1992; Xiao and Tates, 1994; Lahdetie et al., 1994; Russo et al., 1994). In general, the spermatid micronucleus assays with rats and mice indicated that acryl- amide induced most of its effect in primary sper- matocytes at premeiotic Gl-S phases. Some ef- fect was also noted in late spermatogonial stages and in primary spermatocytes at leptotene and zygotene. In mice, a similar induced response for micronuclei was seen in spermatids and periph- eral blood reticulocytes from the same animals (Russo et al., 1994). Though these results demon- strate some sensitivity of these germ cell stages to acrylamide clastogenic effects, this activity was reported as weak. Other assays, such as the domi- nant lethal assay, point to the late spermatids/ early spermatozoa stages as the primary sensitive stages to acrylamide exposure. Consistent with this stage sensitivity, a cytogenetic analysis of first cleavage metaphases of one-cell zygotes after acrylamide exposure of male parents’ spermato- zoa and spermatocytes revealed significant in- creases in structural aberrations (Valdivia et al., 1989; Pacchierotti et al., 1994).

In a mouse spermatid micronucleus assay, acrylamide treatment produced kinetochore-posi- tive staining, suggesting a possible aneuploidy effect (Collins et al., 1992). An aneuploidy effect is indirectly supported by synaptonemal complex irregularities seen in germ cells (asynapsis in mei- otic prophase) (Backer et al., 1989).

All dominant lethal studies performed in mice and rats demonstrate an effect from acrylamide

exposure (Shelby et al., 1987; Working et al., 1987; Dobrzyiiska et al., 1990; Ehling and Neuhluser-Klaus, 1992; Fail et al., 1992; Gutier- rez-Espeleta et al., 1992). A positive dominant lethal effect is observed after all routes of admin- istration 6.p. injection, gavage, drinking water, and dermal). The lowest dose used for a domi- nant lethal response is observed following drink- ing water exposure (9.2 mg/kg/day over 20 weeks). The other dominant lethal studies pro- duced positive results in the range of 25-125 mg/kg/day. Consistently, the affected germ cell stages of spermatogenesis were the late sper- matids and early spermatozoa.

The observations from the dominant lethal studies led to the conduct of heritable transloca- tion studies. In addition to the specific-locus as- says, heritable translocation assays can be used to provide quantitative data for a heritable risk as- sessment for humans. In the earlier study by Shelby et al. (1987), mice were exposed for 5 days at 40 or 50 mg acrylamide/kg/day. Adler and co-workers have performed two studies, one with a 5 day exposure at 50 mg acrylamide/kg/day (Adler, 1990) and one with single doses of 50 and 100 mg acrylamide/kg (Adler et al., 1994). All three studies detected increased translocation frequencies.

While the Shelby et al. (1987) study used two dose levels, the doses were close enough that their use for a dose-response evaluation was not considered. The number of translocations from semisterile and sterile males was used in the estimation. The result at 5 X 40 mg/kg will be used in the calculations in section 5; the Shelby et al. data are:

Exposure regime Number of Number of translocations offspring

Historical control 17 8095 5 x 40 mg/kg 39 162 5 x 50 mg/kg 49 125

The Adler (1990) and Adler et al. (1994) stud- ies provide dose-response information. The re- sults from the 50 mg/kg dose and the 5 X 50 mg/kg dosing regime will be used in the calcula- tions found in section 5. In addition, the com-

El L. Dearfield et al. /Mutation Research 330 (1995) 71-99 81

bined data will be sis. The data from

Exposure regime

used for dose-response analy- the two studies are:

Number of Number of translocations offspring

P a n H&=fi-C-NH2 + so‘+:

ACRYLAMIDE persulfate

Historical control 3 8700 50 mg/kg 2 362

100 mg/kg 10 367 5 x 50 mg/kg 23 105

Other genotoxic effects Positive effects were generally observed with

acrylamide exposure in several DNA damage and repair-related assays. A positive result was found in the Bacillus subtilis ret assay (Tsuda et al., 1993). For both acrylamide and glycidamide, a negative result was reported in one in vitro un- scheduled DNA synthesis (UDS) assay with pri- mary rat hepatocytes (Barfknecht et al., 1988; Butterworth et al., 1992). However, other UDS studies using primary rat hepatocytes and human mammary epithelial cells were positive (Barf- knecht et al., 1988; Butterworth et al., 1992). In an in viva/in vitro UDS acrylamide study, nega- tive results were found with rat hepatocytes, but positive in rat spermatocytes (Butterworth et al., 1992). Positive UDS results in mouse germ cells were also found in an all in vivo study (Sega et al., 19901. Acrylamide DNA adducts were de- tected in both mouse testis and mouse liver (Sega et al., 1990). DNA damage induced by acrylamide as detected by alkaline elution was found in mice treated in vivo (Sega and Generoso, 1990).

Both in vitro and in vivo sister chromatid ex- change (SCE) assays detected acrylamide activity in cultured Chinese hamster V79 cells and mouse spleen lymphocytes and mouse splenocytes (Knaap et al., 1988; Backer et al., 1989; Kliger- man et al., 1991; Tsuda et al., 1993). Also, an in vivo SCE analysis in differentiating spermatogo- nia indicated a weak acrylamide effect in the premeiotic stages of spermatogenesis (Russo et al., 1994). Evidence shows that acrylamide in- duces cell transformation in C3H/lOTi, NIH/3T3, and BALB/c-3T3 cells (see previous review; also Tsuda et al., 19931, although one

t

POLYACRYLAMIDE

Fig. 1. Polymerization of a&amide monomer subunits into polyacrylamide.

report did not replicate the C3H/lOT$ cell re- sults (Abernethy and Boreiko, 1987).

Other assays examining germ cells demon- strated a weak positive induction of sperm-head DNA alkylations in mice as well as sperm-head protamine alkylation (Sega et al., 1989). This in- formation could provide insight into the mecha- nism(s) of germ cell genetic toxicity (discussed in section 3). Also, acrylamide produced spermhead abnormalities in mice after a drinking water ex- posure (Sakamoto and Hashimoto, 1986).

3. Acrylamide / glycidamide reactivity

There are three possible reactivities of acryl- amide. The first of these is acrylamide’s radical- mediated polymerization, as utilized in its trans- formation to polyacrylamide (Fig. 1). While this polymerization is best achieved anaerobically, its importance to acrylamide’s genotoxicity is un- known simply because it has not yet been studied. It has been suggested that the radical-mediated mechanism could possibly explain the different genotoxicity results observed between bone mar- row and germ cells because sperm have a lower oxygen tension. However, data from Sega et al. (1989) suggest that direct interaction between the acrylamide monomer and sperm protamine may

82 K L. Dearfield et al. /Mutation Research 330 (1995) 71-99

be responsible for most, if not all, of the effects seen in the dominant lethal studies.

The second type of reactivity with a&amide is ‘Michael reactivity’. Michael, in the 188Os, first described the addition of carbanions to certain cr$-unsaturated chemicals. Acrylamide, with its double bond between the (Y and p carbons (see Fig. 11, is a specific example of one of these cY,/?-unsaturated chemicals. It is now customary in biology to consider only the addition of thiol, hydroxy or amino groups to the Michael acceptor. These reactions should, therefore, formally be described as ‘Michael-type’ reactions.

As would be expected, a marker for the Michael-type reactivity of a chemical is the rapid addition of ‘non-critical’ thiol groups from cys- teine to the acceptor. This reactivity would repre- sent a detoxification pathway unless the alkyla- tion of -SH groups in protein (e.g., protamine) leads to genetic damage through chromosome breakage (Sega et al., 1989, 1990; Sega and Gen- eroso, 1990). Only the acrylamide that survives the ‘thiol barrier’ would be free to react with

I-CARBOXYETHYL ADENINE J-CARBOXYETHYL CYTOSINE

(5 5 nanomoledlng DNA, ,2 8 nanDm&Simg DNA)

W

Ati, AHp

#J H,N N

7.FORMAMIDOETHYL GUANINE N%ARBOXYETHYL ADENINE

(1 6 nanmmesimg mw (1 4 “anmalesimg DNA,

I-CARBOXYETHYL GUANINE

(0 3 nanamdeslmg DNA)

Fig. 2. Major alkylation products formed by acrylamide inter- action with DNA.

oxygen

metabolism ii t_ H,G2~C-~_-NH,

H,C=$-C-NH? H

ACRYLAMIDE GLYCIDAMIDE

Fig. 3. Formation of acrylamide epoxide (glycidamide) by oxidation of acrylamide.

DNA in a Michael sense, or be epoxidized to the acrylamide-epoxide electrophile (see below). Mukhtar et al. (1981) found that a single, topical application of acrylamide to mice resulted in a time-dependent depletion in cutaneous and hep- atic glutathione content, glutathione S-trans- ferase and aryl hydrocarbon hydroxylase activi- ties. Their data also support the concept of a ‘thiol barrier’ for acrylamide. Given the propen- sity of acrylamide to interact with -SH groups and proteins, one would expect the mode of clastogenicity to be mechanistically different than DNA-related gene mutagenicity.

Acrylamide has been reported to bind to calf thymus DNA in vitro via Michael-type additions (Solomon et al., 1985). The major alkylation products and their abundances are shown in Fig. 2. While DNA adducts were detected, it required an exposure time of 40 days. Thus, the acryl- amide adducts formed in DNA per unit time were about 2 orders of magnitude lower than the number formed with equimolar concentrations of diethylsulfate, ethylmethanesulfonate, or ethylni- trosourea (Sun and Singer, 197.5).

The third reaction mechanism involves the oxi- dation of the acrylamide double bond to an epox- ide (glycidamide; see Fig. 3). At the present time it is not known if the P450 found in the mam- malian testis can catalyze the oxidation of acryl- amide to glycidamide. However, it is also possible that acrylamide is metabolized elsewhere, e.g., liver, with glycidamide then being transported back to the germ cells. The Segerback et al. report (in press; see below for discussion) demon- strates that glycidamide is a relatively stable epox- ide with a long half-life in vivo and is relatively evenly distributed among tissues. Additionally, the hemoglobin adducts described by Calleman et al. (1990) and the presence of glycidamide in urine (Sumner et al., 1992) (both reports dis-

K. L. Dearfield et al. /Mutation Research 330 (1995) 71-99 83

cussed below) provide further evidence for the stability and distribution of glycidamide.

The repeated observation that acrylamide is non-mutagenic to Salmonella in the presence of S9 mix (Lijinsky and Andrews, 1980; Zeiger et al., 1987; Knaap et al., 1988; Tsuda et al., 1993) is somewhat unusual because Aroclor-induced S9 mix is competent in the epoxidation of a wide range of other olefins (e.g., vinyl chloride, butadi- ene) and polycyclic aromatic hydrocarbons (PAHs) such as dimethylbenzo[ alanthracene (DMBA) and benzo[a]pyrene (BP). It may be possible that a high specificity for a particular form of P450 is necessary for the epoxidation of acrylamide. Furthermore, not all isolated eth- ylenes are mutagenic to Salmonella in the pres- ence of S9 mix. Key examples of non-mutagenic ethylenes are safrole and diallylphthalate. Also, a report by Tanii and Hashimoto (1981) indicated that they could not detect metabolism of acryl-

OXyge”

+

metabolism 0 P

H$=$-C-NHZ e H,C/--\;-C-NH,

ACRYLAMIDE GLYCIDAMIDE

‘&ONH?

guanine GLYCIDAMIDE guanine

ADDUCT-

Y20H ,CH-CONHp

guanine GLYCIDAMIDE guanlne

ADDUCT-

Fig. 4. An example of how two distinct adducts of guanine could be formed by glycidamide depending on which of the two epoxide bonds becomes reactive.

m31 WI

Fig. 5. Metabolites of a&amide found in the urine of rats and mice.

amide in a mouse hepatic assay system without or with phenobarbital pretreatment, while a number of related compounds were metabolized. On the other hand, direct exposure to glycidamide has been reported to be mutagenic in Salmonella in the absence of S9 mix (Hashimoto and Tanii, 1985); this suggests a direct DNA activity by glycidamide.

In summary, glycidamide is mutagenic to Salmonella and would be expected to be gener- ally mutagenic in other assay systems. The only question is whether glycidamide is formed in those situations where a mutagenic response has been reported for acrylamide. Clearly the isolation of DNA adducts would help answer this question as unique adducts would be formed in the manner noted below, using alkylation of the N7 position of guanine as an example (Fig. 4). It should be noted that the in vitro exposures of acrylamide to DNA (Solomon et al., 1985) did not produce either the type-l or type-2 adducts shown in Fig. 4.

Calleman et al. (1990) and Bergmark et al. (1991) used gas chromatography-mass spectrome- try (GC-MS) to identify both S-(Zcarboxyethyl)- cysteine and S-(2-carboxy-2-hydroxyethyl)cysteine

84 R L. Dearfield et al. /Mutation Research 330 (I 995) 71-99

in hydrolyzed hemoglobin samples from rats treated with acrylamide in vivo and in microsomal suspensions of acrylamide with cysteine in vitro. The S-(2-carboxyethyl)cysteine is the hydrolysis product of S-(2-formamidoethyl)cysteine, which is formed by direct alkylation of the hemoglobin by acrylamide. On the other hand, S-(2-carboxy- 2-hydroxyethyl)cysteine is the hydrolysis product of S-(2-formamido-2_hydroxyethyl)cysteine, which is formed by the reaction of glycidamide (acrylamide-epoxide) with the hemoglobin. The latter adduct corresponds to ADDUCT- shown in Fig. 4; the amine group is lost by hydrolysis.

Sumner et al. (1992) used nuclear magnetic resonance to characterize urinary metabolites of acrylamide in mice and rats. The metabolites assigned in rat and mouse urine are shown in Fig. 5 and were N-acetyl-S-(3-amino-3-oxopropylkys- teine [#3], N-acetyl-S-(3-amino-2-hydroxy-3- oxopropyl)cysteine [#ll, N-acetyl-W-carbamoyl- 2-hydroxyethyl)cysteine [#2], glycidamide, and 2,3-dihydroxypropionamide [#4]. (GS represents glutathione.) The authors reported that 70% of the rat metabolites and 40% of the mouse metabolites resulted from direct conjugation of acrylamide with glutathione. Adduct #3 was also found to be the major urinary metabolite in rats exposed to acrylamide in a studies carried out by Miller et al. (1982) and Langvardt et al. (1979).

In studies of hemoglobin adducts formed in the rat by exposure to acrylamide and its metabo- lite, glycidamide, carried out by Bergmark et al. (19911, it was estimated that the percentage of acrylamide converted to glycidamide in the rat decreased from 51% following administration of 5 mg/kg to 13% after a dose of 100 mg/kg. Subchronic treatment of rats with acrylamide (10 mg/kg/day for 10 days or 3.3 mg/kg/day for 30 days) confirmed that the conversion rate of acryl- amide to glycidamide, as determined by hemoglobin adduct formation, is higher at lower administered doses. Thus, dose-rate effects may significantly affect risk estimates for acrylamide. In a subsequent paper (Callernan et al., 19921, it was estimated that the percentage of acrylamide converted to glycidamide approaches 58% as the initial concentration of acrylamide approaches zero.

A recent study by Bergmark et al. (1993) inves- tigated hemoglobin adducts in Chinese workers exposed to acrylamide. GC-MS analysis of the N-terminal valines in hemoglobin detected adducts from both acrylamide and its epoxide metabolite (glycidamide). There was a linear rela- tionship between the acrylamide and glycidamide adduct levels and the ratio of the in vivo doses of glycidamide and acrylamide was 3:lO. This study is important in that it demonstrates that humans, as well as mice and rats, are able to metabolize acrylamide to its epoxide (glycidamide). The study also shows that adducts can be detected in ex- posed humans.

Besides forming adducts with hemoglobin, acrylamide has also been shown to interact strongly with mouse sperm protamines. In terms of establishing genotoxic risk, the interaction of acrylamide with germ cell protamines appears to have an important role. Protamines are low molecular-weight basic proteins which replace histones in the chromosomes of mid-to-late sper- matid stages of most mammals, including mice and men.

In the study by Sega et al. (19891, 14C-labeled a&amide (12.5 mg/kg) was injected i.p. into mice, after which spermatozoa were analyzed daily for 3 weeks. At each time point, alkylation of protamines accounted for the vast majority of sperm-head radiolabel. In late spermatids and early spermatozoa, more than 99% of the radiola- be1 was associated with the protamine. The levels of “C radioactivity bound to DNA did not change with time, while alkylation of protamines peaked ( > lo-fold increase) during the second week after treatment with acrylamide, which coincides with exposure of late spermatids and early spermato- zoa.

Analysis of radiolabeled protamine hydro- lysates showed that cysteine was a major target for acrylamide binding. The primary adduct found was S-(2-carboxyethyl)cysteine, the hydrolysis product of S-(Zformamidoethyl)cysteine, which would be formed by the direct alkylation of the protamine by acrylamide. A smaller, unidentified peak of radioactivity was also observed in the protamine hydrolysates. It is possible this second, smaller adduct peak may have been S-(Zcarboxy-

KL. Dearfield et al. /Mutation Research 330 (1995) 71-99 8.5

2_hydroxyethyl)cysteine, the hydrolysis product of S-(Zformamido-2-hydroxyethyl)cysteine (analo- gous to ADDUCT- in Fig. 4), which would be formed by the reaction of glycidamide with the protamine. In any event, the predominant adduct found in the protamine resulted from the direct interaction of acrylamide with the sulfur groups of cysteine contained in the protamine.

The spermiogenic pattern of protamine alkyla- tion seen by Sega et al. (1989) parallels the germ cell stage sensitivity of rodents to dominant lethal mutations (Shelby et al., 1986; Working et al., 1987), heritable translocations (Shelby et al., 1987) specific-locus mutations (Russell et al., 1991) and DNA strand breakage (Sega and Gen- eroso, 1990). In each case, late spermatids and early spermatozoa were the most susceptible to acrylamide-induced damage. Protamine alkyla- tion is thus suggested to play an important role in the genotoxicity of acrylamide (Sega et al., 1989). A mechanism is proposed whereby direct binding of acrylamide to free cysteine sulfhydryls of pro- tamines in early spermatozoa and late spermatids disrupts normal chromatin condensation, causing stresses in chromatin structure leading to DNA breaks and dominant lethal effects.

Acrylamide treatment of mammalian cells causes other responses which are associated with DNA damage, and which can be detected by appropriate assays. One important assay relevant to DNA adduct formation is the detection of unscheduled DNA synthesis CUDS) in non-repli- cating cells. The detection of UDS indicates that there has been damage to DNA followed by DNA repair as measured by the unscheduled uptake of [3H]deoxythymidine (L3H]dT) into DNA.

UDS was induced by acrylamide treatment in F344 rat pachytene spermatocytes in a gavage study (Hurtt et al., 1987). Spermatogenic cells were isolated and incubated with 13H]dT 4 h after completion of a 5-day treatment with 30 mg/kg/day acrylamide. Twenty-four hours later, autoradiographic analysis showed 6.5 net grains (NG) per spermatocyte nucleus, with 56% of the cells responding (CR), compared to 0 NG and 5% CR in controls.

Hepatocellular DNA repair was induced in

cultured hepatocytes exposed to 30 mM acryl- amide. Barfknecht et al. (1987) found a NG count of 5.3 f 8, with 47% of the cells being in repair. Mast et al. (1983) also observed repair with doses up to 100 mg acrylamide/ml. However, Miller and McQueen (1986) could not detect a UDS response in rat hepatocytes up to exposures that were cytotoxic. Similarly, Butterworth et al. (1992) found that acrylamide did not induce DNA repair in either the in vitro or the in vivo hepatocyte DNA repair assays. This group also reported that glycidamide did induce DNA repair in the in vitro hepatocyte DNA repair assay. However, the NG counts they observed with the two concentra- tions of glycidamide that gave a positive response (1 and 10 mM) were identical at both concentra- tions (4.9 NG). The lack of a dose-response effect raises some questions about dose-related effects.

Following i.p. injection of mice with 125 mg acrylamide/kg and testicular injection of [3H]dT at various times after acrylamide exposure, Sega et al. (1990) measured the UDS response induced in early spermatids. Of note, the maximum UDS response was observed 6 h after the i.p. exposure to acrylamide and was about 5 times greater than the response measured immediately after treat- ment. This is the longest delay between chemical treatment and maximum UDS response yet ob- served in mouse germ cells. Also, a linear rela- tionship between the UDS response and acryl- amide exposure from 7.8 to 125 mg/kg was ob- served. By using 14C-labeled acrylamide, it was determined that the temporal pattern of adduct formation in testes DNA paralleled that of the UDS response, with maximum binding occurring 4-6 h after exposure. In contrast, the temporal pattern of adduct formation in liver DNA showed maximum binding within l-2 h after exposure and was an order of magnitude greater than that found for the testes DNA. In view of the high level of DNA adduct formation in the liver, the negative UDS results with hepatocytes cited above are puzzling.

Segerback et al. (in press) have recently stud- ied DNA adduct formation in vivo in mice and rats following i.p. administration of 14C-labeled acrylamide. The predominant adduct found in both species was N-7-(2-carbamoyl-2-hydroxy-

86 EL L. Dearfield et al. /Mutation Research 330 (1995) 71-99

ethyljguanine (ADDUCT- in Fig. 4) and was detected in liver (rat and mouse) and in kidney, spleen, brain, and testis (these tissues only exam- ined in the rat). This result gives support to the idea that glycidamide is the compound that is reactive with DNA in vivo and not acrylamide itself. Their data are also consistent with the UDS results found by Sega et al. (1990) in mouse germ cells, where the maximum UDS response was observed 6 h after the i.p. exposure to acryl- amide. As proposed by Sega et al. (19901, the extraordinarily long delay in the maximum UDS response in the germ cells was most likely due to the slow conversion of acrylamide to glycidamide which could then react with the weak nucle- ophilic sites in DNA (as compared to the much more nucleophilic -SH groups in proteins that are able to react directly with acrylamide). Whether the site of conversion of acrylamide to glycidamide is in the germ cells themselves or acrylamide is converted elsewhere and then transported back to the germ cells is at present unknown. It should also be noted that unpub- lished studies by Sega showed that in vivo expo- sures of male mice to glycidamide resulted in a UDS response that did not require any waiting period before being detected.

Table 3

Acrylamide versus glycidamide adducts

4. Discussion of possible mechanisms for acryl- amide effects

It has become increasingly clear that acryl- amide has different reactivities that can con- tribute to its mechanisms and effects. Not only does the parent compound, monomeric acryl- amide, have activity via its Michael-type reac- tions, but its conversion product, the acrylamide- epoxide glycidamide, also has biological activity via direct nucleophilic substitution. Several stud- ies have demonstrated the in vivo conversion of acrylamide to glycidamide in rodents and humans (discussed in section 3). As summarized in Table 3, several lines of evidence suggest that acryl- amide’s principal biological activity is due primar- ily to adduction with proteins. It is further sug- gested that the primary activity due to alkylation of DNA is more likely due to glycidamide inter- action. Therefore, exposure to acrylamide can have several results dependent upon the relative amounts of parent versus epoxide metabolite and their respective activities. (The specific studies are presented and discussed in Table 1 and sec- tions 2 and 3).

Several lines of evidence suggest that the pri- mary activity of acrylamide alkylation is probably

HYPOTHESIS

The primary activity by parent compound, acrylamide (AA), is binding to proteins, via Michael-type reactions.

AA has weak binding to DNA. AA can be converted in vivo to glycidamide (GLY). GLY is much more reactive than AA in binding to DNA. GLY also binds to proteins. Therefore, the primary consequences from AA

would be due to protein binding and from GLY would be due to DNA binding and to protein binding.

AA protein adducts GLY DNA adducts

AA is negative in Salmonella assay GLY is positive in Salmonella assay

Long period needed to detect DNA binding after exposure No delay in UDS after exposure

Delay in maximum UDS after exposure, suggests Predominant adduct in DNA in vivo rodents was

conversion needed GLY DNA adduct Major adduct, as expected for epoxide, is at N-7

of guanine Primary urinary metabolites are glutathione adducts

Detectable hemoglobin adducts

Protamine alkylation parallels germ cell stage sensitivity

in dominant lethal and heritable translocation tests

Synaptonemal complex effects (proteinaceous structure)

Spindle disturbances; kinetochore positive staining

S9 inducer (effects proteins?)

DNA adduct level in vivo similar in several tissues;

GLY evenly distributed

Epoxide known to induce gene mutations;

may contribute to spermatogonia specific-locus mutations

K.L. Dearjield et al. /Mutation Research 330 (1995) 71-99 87

due to protein binding. Early investigations indi- cated a lack of Salmonella positive results. Al- though some DNA binding was shown in an in vitro situation, acrylamide only demonstrated weak activity and the binding took an inordinate length of time to occur. While there were positive results in the dominant lethal and micronucleus assays, there are some alternative mechanisms other than direct DNA interaction that could account for the results, i.e. protein binding via protamine alkylation and aneuploidy effects (spindle disturbances), respectively. Certainly, protamine alkylation parallels the germ cell stages sensitive to acrylamide dominant lethal and heri- table translocation effects. This could account for most of the results seen in these two assays.

In addition, the primary urinary metabolites and hemoglobin adducts are those resulting from direct alkylation by acrylamide to thiol groups in glutathione and hemoglobin. It is noted that there are some detectable glycidamide urinary metabo- lites and hemoglobin adducts. This suggests a possible role for glycidamide effects due to alky- lation of thiol groups in protein in addition to acrylamide’s primary activity. Other evidence from interactions with the synaptonemal complex (a proteinaceous structure) and induction of rat hepatic enzymes (which may affect S9 proteins; Gamal El-Din et al., 1993) indirectly suggests a role for alkylation to proteins by acrylamide.

The DNA alterations seen with acrylamide exposure are more probably due to its epoxide, glycidamide. Glycidamide has been shown to be positive in Salmonella, unlike the parent acryl- amide (it is not clear why the exogenous metabolic

activation systems for Salmonella do not ‘activate’ acrylamide). Once acrylamide is converted to gly- cidamide, several outcomes may be attributed to glycidamide. Since there was a delay in maximum UDS after acrylamide exposure to germ cells, and no delay after glycidamide exposure, the DNA alteration is presumed to be due to glycidamide. It has now been shown that the predominant adduct in rodent DNA in vivo was the N-7 ad- duction of guanine, as expected for an epoxide. Now that spermatogonial specific-locus mutations have been detected (in addition to post-meiotic effects), it is suggested that the DNA activity by glycidamide plays a prominent role for gene mu- tations.

Both acrylamide and glycidamide appear to freely distribute systemically and have access to the germ cell compartments. Labeled acrylamide can still be found in testis several days after initial exposure. While both compounds can react with proteins, form hemoglobin adducts, and have ready access to most target tissues, they differ in their reactivity to DNA. This would have implica- tions for long-term versus short-term reproduc- tive outcomes and quality of sperm. Gene muta- tions in spermatogonia would indicate long-term risk, whereas protamine alkylation in post-meiotic germ cell stages would indicate shorter-term risks. There is a suggestion that glycidamide induces more reproductive toxicity than does acrylamide (decreased testis protein content and epididymis weight, decreased vas deferens sperm count and sperm cell viability; Costa et al., 1992). The exact molecular and cellular mechanisms for all these effects still need to be examined.

In Vitro Somatic in Vivo Mouse Germ Cells

Specific DNA Adducts __) Specific DNA Adducts d Specific DNA Adducts

Measured Mutations - Measured Mutations b Estimated Mutations

Fig. 6. Modification of original parallelogram as described by Sobels (Sobels, 1982,1989).

88 K L. Dearfield et al. /Mutation Research 330 (1995) 71-99

5. Genetic risk approaches

Parallelogram approach As detailed in the previous sections, there

exists a substantial quantitative data base for acrylamide-induced genetic effects. There is also a significant amount of metabolic and mechanis- tic information that can be used to understand the potential for acrylamide to induce adverse effects in both the somatic and germ cells of humans. With such an available data base, acryl- amide was selected for this workshop as a possi- ble candidate for application of the parallelogram approach to quantitative risk assessment.

From the discussion by Wright (this issue), there are several possible parallelograms that can be constructed (see Figs. 6 and 7). Fig. 6 is a modification of the original parallelogram as de- scribed by Sobels (Sobels, 1982, 1989). It incorpo- rates somatic in vivo data into the model. With the current technologies, it would be possible, for example, to obtain the information required to develop the associations between specific DNA adducts and their genetic outcomes. In fact, it would be possible to measure effects in mouse germ cells and to determine if the relationship between specific DNA adducts (or other alter- ations) and outcome is the same in the cells treated in vitro, somatic cells treated in vivo, and germ cells treated in vivo. It would also be possi- ble to determine the relationship between ap- plied dose and specific DNA adduct production. To do such an analysis would require that the endpoint measured in vitro, in somatic cells in

Mouse Human

Somatic Cell a Somatic Cell

Measured potency Measured potency

b

I

Germ Cell a

_) Germ Cell

Measured potency Estimated potency

Fig. 7. Parallelogram relating rodent information to the hu-

man situation (addressing issue of human risk assessment>.

vivo, and in germ cells was comparable. Because the chromosomal endpoints that can be measured are somewhat different in somatic cells and germ cells (see Allen et al., this issue), perhaps it would be best to use a point mutation assay for this type of analysis. However, acrylamide appears to act primarily via a clastogenic mechanism (aneu- ploidy perhaps; via protein (protamine) adduc- tion). While the specific-locus mutations suggest a point mutation mechanism, there are very few other related data to make the parallelogram approach applicable. Furthermore, in spite of the large data base available, no information has been generated relating specific adduct formation to a measured mutational outcome.

The model shown in Fig. 6 does not, however, address the issue of human risk assessment. A parallelogram such as shown in Fig. 7 would be required to relate rodent information to the hu- man situation. For this model, the assumption is made that the mathematical relationship (b) be- tween the somatic cell and the germ cell effect is the same in rodents and humans. It further as- sumes that the mouse-human somatic cell out- come relationship (a) is the same as the mouse- human germ cell outcome relationship. The mea- sured potency would in each case include the dose-effect relationship, and could possibly be determined by using specific DNA adduct forma- tion. As with the model in Fig. 6, it is important that the outcome used in all three of the mea- sured potencies be equivalent. Again, in spite of all the available information on acrylamide ef- fects, this type of specific data is not available for application in the parallelogram model. In partic- ular, there is very little information for acryl- amide interaction with human somatic cells, the hemoglobin adduction in Chinese workers being the main data (Bergmark et al., 1993). There is no comparative endpoint in germ cells to estab- lish similar biological endpoint dosimetry. It should also be noted that this particular parallel- ogram model is not easily validated. The tech- niques to measure potency of effect in human germ cells following chemical exposure are not available.

Thus, although the parallelogram model is at- tractive in its approach, it is not easy to obtain

K.L. Dearjield et al. /Mutation Research 330 (1995) 71-99 89

the quantitative information required to either validate or use it for genetic risk assessment. Furthermore, the parallelogram approach does not provide a means to estimate increased inci- dences of genetic disease(s). Having examined the parallelogram approach, the Workgroup de- termined that while there was insufficient infor- mation to use this approach, enough information was available to use either a direct approach (or modified direct approach) or a doubling dose approach for performing genetic risk assessments. The parallelogram approach was used to identify data that could help refine the extrapolation fac- tors used in the genetic risk calculations (rela- tionships a and b in Fig. 7).

Acrylamide dosimetry and extrapolation factors In order to quantitatively estimate human ge-

netic risk, one must determine the dose of acryl- amide to which humans are exposed. In the mu- tagenesis literature, the dose often equals an exposure of the organism in terms of concentra- tions applied in ppm, mM, mg/kg, etc. and does not represent the amount of chemical or its metabolite that actually reaches the gonads. As pointed out by several authors, the chemical dose would be better defined as the integral of time- dependent concentration (see Ehling et al., 1983; Ehrenberg et al., 1974; Latarjet, 1977):

D = /‘C( t)dt 0

After the establishment of the dose, it is nec- essary to determine the dose-response relation- ship of acrylamide. A dose-response experiment typically includes controls, two, or preferably more, graded doses, replicates, and an experi- mental design which pays careful attention to minimizing confounding factors. Exposure dose, dose rate and route of administration may or may not influence the dose-effect response. In the simplest case, response depends linearly on dose: y - y, = bx, where y is the observed mutations per survivor, y, is the background or spontaneous mutations per survivors, x the dose and b is the slope of the dose-response line. A detailed analy-

sis of dose-response relationships in chemical mutagenesis was given by Ehling et al. (1983).

The importance of the differential spermato- genie response following chemical exposure needs to be considered. The identification of the germ cell stage-specific induction of mutations is essen- tial for risk assessment. Post-spermatogonia go through meiotic divisions and through successive differentiation stages, i.e. spermatocytes, sper- matids, and spermatozoa. Production of a mature sperm from a spermatogonial stem cell (A, sper- matogonia) takes about 5 weeks in the mouse (Oakberg, 1968) and almost twice as long in man (Heller and Clermont, 1964). The spermatozoa, spermatids, and spermatocytes do not persist for long in the body, less than 0.7% of the human generation time. Accordingly, mutations induced in post-spermatogonial stages have only a transi- tory chance of fertilization. In contrast, stem cell spermatogonia are reproduced over the whole reproductive lifespan. Mutations induced by acrylamide in A, spermatogonia are therefore reproduced over the whole reproductive lifespan. For example, a male acutely exposed to acryl- amide would have a greater risk of fathering an affected offspring over his reproductive lifespan if the spermatogonia are mutated versus a post- spermatogonial germ cell. There would be a lim- ited time of risk for fathering an affected off- spring if only post-spermatogonial germ cells were mutated. This should be remembered when com- paring the heritable genetic risk estimates de- rived from the specific-locus test (spermatogonia affected) versus the heritable translocation tests (late spermatids/early spermatozoa affected). For females, the yield of induced mutation rate in oocytes, in general, is lower than in spermatogo- nia. However, to perform a full risk assessment, it would be desirable to have data from both male and female animals.

Using the data from the mouse to arrive at quantitative estimates of the genetic risk to hu- mans, assumptions are made (Ehling, 1988): (1) The amount of genetic damage induced by a

given type of exposure under a given set of conditions is the same in the germ cells of mice and humans.

(2) The various biological and applicational fac-

90 K.L. Dearfield et al. /Mutation Research 330 (1995) 71-99

tors affect the magnitude of the induced mu- tation frequency in similar ways and to similar extents in mice and in humans.

Generally, the parallelogram is useful to help make refinements in the assumptions and extrap- olation factors referred to above. As presented by the ICPEMC Workgroup, several risk extrapola- tion factors (REFsI between rodent (mice) exper- imental models and humans were assumed for acrylamide risk calculations (ICPEMC, 1993a):

Parameter REF

Locus specificity 2 DNA repair variability 0.1 Metabolic variability 1 Dose rate variability 1 Exposure route 1 Germ cell stage specificity 1 Dose-response kinetics 1 Overall REF 0.2

The assumption that the human genome is more ‘mutable’ than the mouse genome is re- flected in the locus specificity parameter. This is based on the human spontaneous rate which ap- pears slightly higher; therefore, the induced mu- tation susceptibility would be higher also. The DNA repair variability reflects the assumption that repair capabilities in humans are more effec- tive than in mouse. To properly define the other parameters, more data related to acrylamide are needed.

It is further assumed for the calculations at the end of this report that any effect seen in germ cells is an integration of parent acrylamide and its metabolite glycidamide. Evidence to support this assumption was presented at the conference (Generoso, communication in USEPA, 1994) where glycidamide was reported to be an effec- tive inducer of dominant lethal mutations with the same germ cell stage sensitivity (post-meiotic) as acrylamide. Additionally, little difference was observed in the genetic response to equal dose levels of acrylamide and glycidamide.

Direct (modified direct) and doubling dose ap- proaches

Two general approaches were used by the Workgroup to extrapolate mammalian germ cell

acrylamide data to estimate human genetic risk, the direct and the doubling dose approaches (e.g. discussed in more detail in Ehling, 1988; ICPEMC, 1993a,b). It was noted that the ‘direct’ approach actually used a recessive mutation rate to predict dominant diseases; therefore, it was not a true direct approach (i.e., not using a domi- nant endpoint as dominant skeletal or cataract alterations). The Workgroup decided to refer to this approach as the ‘modified’ direct approach.

It should be kept in mind that the approaches used in the modified direct and doubling dose calculations will be used to determine the num- ber of new genetic diseases due to the induction of dominant disease phenotypes. It is recognized that genetic germ cell risk also can be due to the induction of recessive mutations. An assumption being made is that an equal number of mutations in genes causing recessive diseases will be in- duced in the first generation (ICPEMC, 1993a,b). The effects of these recessive mutations will not be expressed in the first generation, but will be important in the risk to subsequent generations. However, these recessive mutations are not fac- tored into the following equations for dominant phenotypes and hence the overall risk will likely be underestimated for future generations.

Modified direct approach. The modified direct approach requires a per locus mutation rate esti- mated in the mouse as well as an estimate of the number of loci in humans capable of mutating to dominantly expressed disease alleles. This num- ber of human loci is critical in the estimation of risk to exposed humans. However, this number is not precisely known at this time and an assump- tion is used. For dominant single gene diseases, the number of human loci was set at 1000 for purposes of deriving estimates of risks; for domi- nant chromosomal diseases, the number was set at 10. Both of these numbers are subject to fur- ther discussion and refinement. With this in mind, the following approach was used to estimate the number of new diseases in the offspring using the modified direct approach (ICPEMC, 1993a,b):

Number of new diseases in the offspring

= REF x M,,,,, x Lhuman x D x N

R L. Dearfield et al. /Mutation Research 330 (1995) 71-99 91

where: M ,,,OuSe = induced per locus mutation rate per unit dose exposure estimated in the mouse; L human = number of loci in humans at which dom- inant disease mutation may arise; D = exposure dose; N = number of offspring descendent from exposed parents; REF = risk extrapolation factor (see above for acrylamide).

Doubling dose approach. The doubling dose approach does not require a specific estimate of the number of human loci which mutate to domi- nant disease alleles as does the modified direct approach. The doubling dose approach does re- quire an estimate of the overall spontaneous mu- tation frequency in humans to dominant disease alleles. These data are usually more easily avail- able thus making this approach preferable to the modified direct approach. In particular, there is greater uncertainty about the number of disease- associated loci in humans than about the sponta- neous mutation rate in humans. For an estimate of the spontaneous mutation rate, the estimate from the UNSCEAR effort is used: 1.5 X lop3 (UNSCEAR, 1986). An estimate of the sponta- neous chromosomal aberration rate is from Sankaranarayanan (1982): 6.2 X 10p8. These mu- tation frequencies in humans were used in the following approach to estimate the number of new diseases in the offspring using the doubling dose approach (ICPEMC, 1993a,b):

Number of new diseases in the offspring

= REF X Sponhumans X D/DD X N

where: Spon humans = overall spontaneous muta- tion rate to dominant disease alleles in humans; D = exposure dose; DD = doubling dose esti- mated in the mouse (that dose which induces a mutation rate equal to the spontaneous mutation rate). The DD is calculated as the mouse sponta- neous rate per unit dose; N = number of off- spring descendent from exposed parents; REF = risk extrapolation factor (see above for acryl- amide).

In the determination of the doubling dose (DDI, a major assumption of linearity is made. The gene mutation data are based on a single data point and no other dose-response informa- tion is available to suggest a non-linear response to model. For chromosomal effects, linearity may

be a reasonable assumption. From an empirical examination of acrylamide data at doses of 100 mg/kg and lower, most of the data from the dominant lethal studies have a linear component (e.g., the dermal dominant lethal study). Also, the Adler et al. (1994) data from the control and the 50 and 100 mg/kg doses can be fitted to a linear equation. The DD calculations from the four major studies are based on the following calcula- tion:

Spontaneous mutation rate DD=

Induced mutation rate/unit exposure

From the Ehling and Neuhauser-Klaus (1992) data:

DD =

22/248 413

[ (6/23 489) - (22/248 413)] /lOO mg/kg

= 53.1 mg/kg

From the Shelby et al. (1987) data:

17/8095

DD = [(39/162) - (17/8095)]/200mg/kg

= 1.8mg/kg

From the Adler et al. (1994) data:

3/8700

DD = [ (2/362) - (3/8700)] /50 mg/kg

= 3.3 mg/kg

From the Adler (1990) data:

3/8700

DD = [(23/105) - (3/8700)]/250mg/kg

= 0.39 mg/kg

While the above calculations were performed using a linear assumption, the data combined from both of the Adler group reports also can be analyzed using a fitted dose-response curve of the Weibull type (Adler et al., 1994). A Weibull-type curve fitting may be consistent with the mecha- nism of translocation production (discussed in Rhomberg et al., 1990). Statistical independence of induced response from the background fre- quency was assumed. Independence is assumed since the background frequency is determined to

92 K L. Dearfield et al. /Mutation Research 330 (1995) 71-99

be a rare event and the occurrence of a chemi- cally induced break interacting with a sponta- neous break in the production of a translocation would be extremely rare, especially at low doses. The DD estimate obtained by maximum likeli- hood calculations based on a human background translocation frequency of 1.9 per 1000 newborns (Lyon et al., 1983) is about 25 mg/kg (Adler et al., 1994).

6. Human exposure

Human exposure to acrylamide may occur by inhalation, ingestion, and skin absorption (for current review, see IARC monograph on acryl- amide, in press). Inhalation exposures may occur during the manufacture of monomer and the use of dry powder or crystalline monomer in the production of polyacrylamide. The largest use of polyacrylamides is as a flocculent in the clarifica- tion of potable water and treatment of wastewa- ter. This provides a high potential for exposure of millions of people to unreacted monomer through ingestion in drinking water. Use of polyacryl- amide grouts comprises the major use of acryl- amide monomer itself. Dermal exposure to monomer would occur mainly via preparation of polyacrylamide solutions as in grouting opera- tions. The human exposure numbers identified by USA regulatory agencies (USEPA, U.S. Occupa- tional Safety and Health Administration (OSHA), U.S. Food and Drug Administration (USFDA)) will be the main source of exposure data used in the genetic risk assessment (section 7).

The Workgroup decided to base the cumulative human exposures on daily values, e.g., an 8-h workday for occupational exposures. The human exposure numbers detailed in this section have been converted to mg/kg-day values (ICPEMC, 1993a).

Inhalation exposure The current OSHA permissible exposure limit

(PEL) in workplace air is 0.3 mg/m3 (8-h time weighted average (TWA)) for acrylamide mono- mer inhalation (OSHA, 1989, 1993). In the OSHA 1989 rule for acrylamide, OSHA published a limit

of 0.03 mg/m3, but this final rule limit was re- voked in response to the 11th Circuit Court of Appeals decision (AFLCIO v. OSHA) effective June 30, 1993; therefore, the PEL reverted back to the original 0.3 mg/m3 limit. The 0.03 mg/m3 limit is the threshold limit value (TLV) published by the American Conference of Governmental Industrial Hygienists (ACGIH, 1994). In a manu- facturing factory in the People’s Republic of China, it has been reported that air exposures ranged in the summer months from 0.3 to 8.8 mg/m3 (Bergmark et al., 1993; Calleman et al., 1994). In an assessment of airborne exposure to acrylamide during chemical grouting operations in the United States, the USEPA sampling of line maintenance workers found 8-h TWA exposures of 0.008-0.12 mg/m3 (USEPA, 1987). These lat- ter exposure numbers are the values used in the representative assessments for acrylamide. These estimates assume 100% absorption for acrylamide via inhalation.

The daily inhalation exposure for a 75 kg per- son inhaling 14 l/min of acrylamide-con- taminated air (Layton, 1993) during an 8-h work period would be:

xmg 141 m3 480min 1

m3 -Xx

XminX 10001 day X-

75 kg

ymg =-

kg-day

Daily inhalation values derived from this calcula- tion are: From OSHA PEL: 0.027 mg/kg-day

From grout workers, lower value: 0.00072 mg/kg-day From grout workers, upper value: 0.011 mg/kg-day From manufacturing, upper value: 0.79 mg/kg-day

(Chinese manufacturing plant)

Ingestion exposure The USFDA limited the amount of residual

acrylamide monomer in polyacrylamide used as a boiler water additive in food processing to not exceed 0.05% by weight of acrylamide monomer (USFDA, 1994). Also, any acrylamide associated resins used in food (secondary direct food addi- tives) cannot contain more than 0.05% residual acrylamide (USFDA, 1994). The USEPA adopted

KL. Dearfield et al. /Mutation Research 330 (1995) 71-99 93

a similar approach for limiting the amount of acrylamide used for water treatment flocculents. Because there are no standard analytical methods available for acrylamide at low levels in drinking water, the USEPA proposed a treatment tech- nique requirement instead of a maximum con- taminant level (MCL); the MCL would be im- practical to enforce. The level for acrylamide is 0.05% acrylamide in polyacrylamide dosed at 1 ppm (USEPA, 1991). If it is assumed that 1 mg/l= 1 ppm, then the limit for acrylamide monomer would be 0.5 pg/l. This limit value will be used in the ingestion (drinking water) assess- ment for acrylamide. Indications from studies suggest that acrylamide absorption from the gas- trointestinal tract is rapid and complete following oral exposure and does not depend upon dose or concentration (e.g., see Miller et al., 1982). This assessment assumes 100% absorption via inges- tion.

The daily ingestion exposure for a 75 kg per- son ingesting 2 liters of water a day with residual acrylamide monomer would be:

xmg 21 1 ymg _~_~----_- 1 day 75 kg kg-day

Daily ingestion value derived from this calcula- tion is:

From USEPA requirement:

1.3 x lop5 mg/kg-day

Dermal exposure Dermal exposure to monomer generally occurs

via preparation of polyacrylamide solutions. One major dermal exposure scenario deals with sewer line maintenance procedures where monomeric acrylamide is mixed into polyacrylamide solutions for use as grouts to seal sewer lines. Another is the use in the research setting for the preparation of polyacrylamide gels. In setting the limits for air contaminants, OSHA also gave acrylamide a skin designation indicating absorption via skin could be of concern (OSHA, 1993). The USEPA con-

ducted an assessment of dermal contact during chemical grouting operations in the United States and found dermal contacts estimated at 0.61-5.0 mg/h (USEPA, 1987). It is assumed that about 25% of the dermal contact dose will be absorbed via the skin (Ramsey et al., 1984 (in Dearfield et al., 1988); Frantz et al., 1985).

The daily skin exposure for a 75 kg person exposed to acrylamide (assuming 25% skin ab- sorption) during an 8-h work period would be:

xmg 8h 1 ymg -x-x---

h day 75 kg x0.25= -

kg-day

Daily dermal values derived from this calculation are:

From grout workers, lower value: From grout workers, upper value:

0.016 mg/kg-day

0.13 mg/kg-day

7. Assessment of heritable genetic risk for hu- mans

The animal germ cell test results can be com- bined with the human exposure figures to obtain an estimate of heritable genetic risk to humans exposed to acrylamide. The risk calculations for the modified direct and doubling dose ap- proaches are used to derive estimates of the frequency of dominant genetic disease burden in the offspring of males exposed to a specified dose of acrylamide. Specific genetic diseases due to acrylamide exposure cannot be predicted at this time.

The estimated number of new (induced) ge- netic diseases due to acrylamide exposure is found in Table 4. The N in each of the risk calculations is set at one million (1 X 106); the number of new genetic diseases is therefore per million offspring. The risk extrapolation factor (REF) is 0.2 as discussed in section 5. An example calculation for

P

Tab

le 4

H

erita

ble

gene

tic

risk

est

imat

es

for

hum

ans

expo

sed

to a

cryl

amid

e

End

poin

t M

ouse

dos

e (m

g/kg

) A

ppro

ach

Dou

blin

g do

se

Num

ber

of i

nduc

ed

gene

tic

dise

ases

pe

r m

illio

n of

fspr

ing

(dos

e sc

hedu

le)

(DD

) (m

g/kg

)

Inge

stio

n In

hala

tion

Der

mal

1.3x

10-

5 0.

027

0.00

072

0.01

1 0.

016

0.13

m

g/kg

-day

m

g/kg

-day

ru

g/kg

-day

m

g/kg

-day

m

g/kg

-day

m

g/kg

-day

O

SHA

PE

L

grou

t w

orke

r gr

out

wor

ker

grou

t w

orke

r gr

out

wor

ker

Gen

e m

utat

ion

100

a (s

ingl

e)

Dou

blin

g do

se

53.1

7.

3x

10-5

0.

15

0.00

4 0.

062

0.09

0.

73

Mod

ifie

d di

rect

4.

3x10

-3

9.0

0.24

3.

7 5.

3 43

.4

Chr

omos

omal

20

0 b(

Sx40

) D

oubl

ing

dose

1.

8 3.

0x10

-*

6.3

0.17

2.

6 3.

7 30

.3

alte

ratio

ns

Mod

ifie

d di

rect

3.

1x10

-2

64.4

1.

7 26

.3

38.2

31

0 50

c (

sing

le)

Dou

blin

g do

se

3.3

1.7x

10

-3

3.4

0.09

1.

4 2.

0 16

.5

Mod

ifie

d di

rect

2.

7 x

1O-3

6.

0 0.

15

2.3

3.3

27.0

25

0 d

(5 x

50)

D

oubl

ing

dose

0.

39

1.4x

10

-2

29.1

0.

78

11.8

11

.2

140

Mod

ifie

d di

rect

2.

3 x

lo-’

47

.2

1.3

19.2

28

.0

227

Com

bine

d ‘z

d D

oubl

ing

dose

25

2.

2x

10-4

0.

45

0.01

0.

18

0.27

2.

2

a E

hlin

g an

d N

euhi

iuse

r-K

laus

(1

992)

. b

Shel

by e

t al

. (1

987)

. ’

Adl

er

et a

l. (1

994)

. d

Adl

er

(199

0).

F

KL. Dearfield et al. /Mutation Research 330 (1995) 71-99 95

gene mutations via ingestion by the modified direct approach is:

Number of new diseases in the offspring

= REF x M mOuSe X L human X D X N

=0.2x [(6/23489) - (22/248413)]

100

x 1000 x (1.3 x 10-5) x (1 x 106)

= 4.3 x 10e3 new dominant

diseases due to gene mutation per million

offspring

An example calculation for gene mutations via ingestion by the doubling dose approach is:

Number of new diseases in the offspring

= REF X Sponhuman X D/DD X N

= 0.2 x (1.5 x 10-j) x (1.3 x 105)

53.1

x (1 x 106) = 7.3 X lop5 new dominant

diseases due to gene mutation per million

offspring

These risk estimates provide an indication of potentially induced genetic diseases after expo- sure to acrylamide. For example, the range for number of new genetic diseases after ingestion exposure for either gene mutations or chromoso- ma1 alterations determined by the doubling dose and modified direct methods is 7.3 x 10P5 to 3.1 x 10e2 induced genetic diseases per million offspring (i.e., up to 0.031 offspring potentially affected per million offspring or about 3 offspring with induced genetic disease per 10’ offspring). Since millions of people are potentially exposed to acrylamide via drinking water, it is useful to know that the projected risk appears less than that seen in occupational settings via inhalation or dermal exposures. From the estimates in Table 4, a greater risk is suggested for affected off- spring from fathers exposed at the upper range of acrylamide concentrations during grouting work. However, when comparing these numbers, the exposure scenario also should be assessed, e.g., grout work is performed by a small number of

workers during a limited number of days per year whereas millions of people are potentially ex- posed to low levels of acrylamide on a more continuous basis via drinking water.

Not only do the exposure scenarios need to be assessed, but the germ cell targets need to be taken into account for the assessment. In deriving these estimates, the human exposures were ex- pressed as mg/kg per day (i.e. equivalent to an 8-h working day for occupational exposures). While humans probably would not be exposed every day of their reproductive life, any mutations induced in spermatogonial stem cells could result in potential adverse outcomes (increased risk) for the remainder of their reproductive life. Due to the nature of the chromosomal alterations stud- ied, these effects are observed mostly during the post-meiotic stages of the germ cell cycle. This would not lead to a continued risk due to chro- mosomal alterations if the exposure was discon- tinued; risk would be increased during the time frame when affected sperm are utilized, i.e., be- fore they are reabsorbed and replaced by unaf- fected sperm (see Rhomberg et al., 1990 for ra- tionale). However, if exposure was continuous, then germ cells moving through the affected post-meiotic germ cell stage(s) would continually have the opportunity for damage (via chromoso- ma1 alterations) and thus contribute to a sus- tained increased risk.

It is recognized that dominant genetic diseases can be induced by both gene mutations and chro- mosomal alterations. However, the risks from these two endpoints are presented separately. It is not clear how the two estimated risks should be combined into a single risk number at this time. Furthermore, these risk estimates are for exposed males; there need to be additional studies to estimate risks from exposed females.

The values used for spontaneous mutation rates for dominant disease alleles in humans and for number of loci in humans at which dominant disease mutations may arise are only general esti- mates. As research sheds more light upon these estimates, the risk calculations themselves will become more reliable. At this time there is more confidence in the estimates for the doubling dose approach than for the modified direct approach.

96 RL. Dearfiild et al. /Mutation Research 330 (1995) 71-99

The doubling dose can be defined as the dose necessary to induce as many mutations as occur spontaneously in one generation. The doubling dose can be used to estimate the individual risk of an exposed person. Furthermore, if the popu- lation dose can be determined, then the genetic risk of the compound for a given exposed popula- tion can be estimated.

Acknowledgements

The Workgroup would like to acknowledge the assistance and input from several individuals dur- ing this effort: Mike Waters and Canice Nolan for organizing the workshop and gathering back- ground materials, the ICPEMC taskgroup (par- ticularly Heinrich Malling and Jack Favor for their additional comments), Ilse-Dore Adler and Waldy Generoso for their keen interest in the acrylamide assessment, and Fred de Serres for his continued support for advancing genetic risk assessment.

References

Abernethy, D. and C. Boreiko (19871 Acrylonitrile and acryl- amide fail to transform C3H/lOTi cells, Environ. Muta- gen., 9 (Suppl. 81, 2.

ACGIH (American Conference of Governmental Industrial Hygienists) (1994) 1993-1994 Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Limits, Cincinnati, OH.

Adler, I.-D. (1990) Clastogenic effects of acrylamide in differ- ent germ-cell stages of male mice, in: J. Allen, B. Bridges, M. Lyon, M. Moses and L. Russell (Eds.1, Biology of Mammalian Germ Cell Mutagenesis, Banbuty Report Vol. 34, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 115-131.

Adler, I.-D., I. Ingwersen, U. Kliesch and A. El Tarras (19881 Clastogenic effects of acrylamide in mouse bone marrow cells, Mutation Res., 206, 379-385.

Adler, I.-D., R. Zouh and E. Schmid (1993) Perturbation of cell division by acrylamide in vitro and in vivo, Mutation Res., 301, 249-254.

Adler, I.-D., P.. Reitmer, R. Schmiiller and G. Schriever- Schwemmer (1994) Dose-response for heritable transloca- tions induced by acrylamide in spermatids of mice, Muta- tion Res., 309, 285-291.

Backer, L., K. Dearfield, G. Erexson, J. Campbell, B. West- brook-Collins and J. Allen (1989) The effects of acryl-

amide on mouse germ-line and somatic cell chromosomes, Environ. Mol. Mutagen., 13, 218-226.

Barfknecht, T., D. Mecca and R. Naismith (19871 Evaluation of acrylamide in rodent hepatocyte DNA/repair assays, Environ. Mutagen., 9 (Suppl. 81, 10-11.

Bartknecht, T., D. Mecca and R. Naismith (1988) The geno- toxic activity of acrylamide, Environ. Mol. Mutagen., 11 (Suppl. 111, 9.

Batiste-Alentorn, M., N. Xamena, A. Creus and R. Marcos (1991) Genotoxicity studies with the unstable Zeste-White (UZ) system of Drosophila melanogaster: results with ten carcinogenic compounds, Environ. Mol. Mutagen., 18, 120-125.

Bergmark, E., C. Calleman and L. Costa (1991) Formation of hemoglobin adducts of acrylamide and its epoxide metabo- lite glycidamide in the rat, Toxicol. Appl. Pharmacol., 111, 352-363.

Bergmark, E., C. Calleman, F. He and L. Costa (19931 Deter- mination of hemoglobin adducts in humans occupationally exposed to acrylamide, Toxicol. Appl. Pharmacol., 120, 45-54.

Butterworth, B., S. Eldridge, C. Sprankle, P. Working, K. Bentley and M. Hurtt (1992) Tissue-specific genotoxic effects of actylamide and acrylonitrile, Environ. Mol. Mu- tagen., 20, 148-155.

Calleman, C., E. Bergmark and L. Costa (1990) Acrylamide is metabolized to glycidamide in the rat: evidence from hemoglobin adduct formation, Chem. Res. Toxicol., 3, 406-412.

Calleman, C., L. Stern, E. Bergmark and L. Costa (1992) Linear versus nonlinear models for hemoglobin adduct formation by acrylamide and its metabolite glycidamide: implications for risk estimation, Cancer Epidemiol. Biomarkers Prevent., 1, 361-368.

Calleman, C., Y. Wu, F. He, G. Tian, E. Bergmark, S. Zhang, H. Deng, Y. Wang, K. Crofton, T. Fennel1 and L. Costa (19941 Relationships between biomarkers of exposure and neurological effects in a group of workers exposed to acrylamide, Toxicol. Appl. Pharmacol., 126, 361-371.

CihLk, R. and M. Vontorkovl (1988) Cytogenetic effects of acrylamide in the bone marrow of mice, Mutation Res., 209, 91-94.

Cihak, R. and M. Vontorkova (1990) Activity of acrylamide in single-, double-, and triple-dose mouse bone marrow mi- cronucleus assays, Mutation Res., 234, 125-127.

Collins, B., D. Howard and J. Allen (1992) Kinechore-staining of spermatid micronuclei: studies of mice treated with X-radiation or acrylamide, Mutation Res., 281, 287-294.

Costa, L., H. Deng, C. Gregotti, L. Manzo, E. Faustman, E. Bergmark and C. Calleman (1992) Comparative studies on the neuro- and reproductive toxicity of acrylamide and its epoxide metabolite glycidamide in the rat, NeuroToxicol- ogy, 13, 219-224.

Dearfield, K., C. Abernathy, M. Ottley, J. Brantner and P. Hayes (1988) Acrylamide: its metabolism, developmental and reproductive effects, genotoxicity, and carcinogenicity, Mutation Res., 195, 45-77.

KL. Dea@eld et al. /Mutation Research 330 (1995) 71-99 91

Dobrzydska, M., M. Lenarczyk and A. Gajewski (1990) Induc- tion of dominant lethal mutations by combined X-ray- acrylamide treatment in male mice, Mutation Res., 232, 209-215.

Ehling, U. (1988) Quantification of the genetic risk of envi- ronmental mutagens, Risk Anal., 8, 45-57.

Ehling, U. and A. Neuhauser-Klaus (1992) Reevaluation of the induction of specific-locus mutations in spermatogonia of the mouse by acrylamide, Mutation Res., 283, 185-191.

Ehling, U., D. Averbeck, P. Cerutti, J. Friedman, H. Greim, A. Kolbye Jr. and M. Mendelsohn (1983) Review of the evidence for the presence or absence of thresholds in the induction of genetic effects by genotoxic chemicals, Muta- tion Res., 123, 281-341.

Ehrenberg, L., K. Hiesche, S. Osterman-Golkar and I. Wennberg (1974) Evaluation of genetic risks of alkylating agents; tissue dose in the mouse from air contaminated with ethylene oxide, Mutation Res., 24, 83-103.

Fail, P., J. George, T. Grizzle, M. Izard, R. Chapin and J. Heindel(l992) Acrylamide (ACRL): reproduction and fer- tility assessment in CD-l mice when administered in drink- ing water, Final study report, NTP/NIEHS Contract No. NOl-ES-65141.

Frantz, S., M. Dryzga, N. Freshour and P. Watanabe (1985) In viva/in vitro determination of cutaneous penetration of residual monomer from polyacrylamides, Toxicologist, 5, 39 (abstr.).

Gamal El-Din, A., H. Al-Maskati, A. Ali Mohamed and M. Dairi (1993) Acrylamide as an inducer of metabolic activa- tion system (S9) in rats, Mutation Res., 300, 91-97.

Gutierrez-Espeleta, G., L. Hughes, W. Piegorsch, M. Shelby and W. Generoso (1992) Acrylamide: dermal exposure produces genetic damage in male mouse germ cells, Fund. Appl. Toxicol., 18, 189-192.

Hashimoto, K. and H. Tanii (1985) Mutagenicity of acryl- amide and its analogues in Salmonella typhimurium, Muta- tion Res., 158, 129-133.

Heller, C. and Y. Clermont (1964) Kinetics of the germinal epithelium in man, in: G. Pincus (Ed.), Recent Progress in Hormone Research, Vol. 20, Academic Press, New York, pp. 545-57s.

Hoom, A., L. Custer, B. Myhr, D. Brusick, J. Gossen and J. Vijg (1993) Detection of chemical mutagens using Muta@Mouse: a transgenic mouse model, Mutagenesis, 8, 7-10.

Hurtt, M., K. Bentley and P. Working (1987) Effects of acrylamide and acrylonitrile on unscheduled DNA synthe- sis (UDS) in rat spermatocytes, Environ. Mutagen., 9, 49-50.

ICPEMC (International Commission for Protection Against Environmental Mutagens and Carcinogens) (1993a) Use of in vivo genetic toxicology data to construct human risk assessments, Final report submitted to Department of Health, Canada, July, 1993, Contract No. 3138.

ICPEMC (International Commission for Protection Against Environmental Mutagens and Carcinogens) (1993b) Ge-

netic risk extrapolation from animal data to human dis- ease, Final report submitted to U.S. Environmental Pro- tection Agency, September, 1993, under interagency agreement number 1824-D094-Al between the USEPA and the Biomedical and Environmental Information Anal- ysis Section of the Oak Ridge National Laboratory.

Kligerman, A., A. Atwater, M. Bryant, G. Erexson, P. Kwanyuen and K. Dearfield (1991) Cytogenetic studies of ethyl acrylate using C57CL/6 mice, Mutagenesis, 6, 137- 141.

Knaap, A., P. Kramers, C. Voogd, W. Be&amp, M. Groot, P. Langebroek, H. Mout, J. van der Stel and H. Verharen (1988) Mutagenic activity of acrylamide in eukaryotic sys- tems but not in bacteria, Mutagenesis, 3, 263-268.

Lihdetie, J., A. Suutari and T. Sjiiblom (1994) The spermatid micronucleus test with the dissection technique detects the germ cell mutagenicity of acrylamide in rat meiotic cells, Mutation Res., 309, 255-262.

Langvardt, P., C. Putzig, J. Young and W. Braun (1979) Isolation and identification of urinary metabolites of vinyl-type compounds: application to metabolites of acryl- onitrile and acrylamide, Toxicol. Appl. Pharmacol., 48, A161.

Latarjet, R. (1977) Quantitative mutagenesis by chemicals and by radiations: prerequisites for the establishment of rad- equivalences, in: R. Chanet (Ed.), Radiobiological Protec- tion. First Eur. Symp. on Rad-equivalences, Commission of the European Communities, Luxembourg, pp. 154-168.

Layton, D. (1993) Metabolically consistent breathing rates for use in dose assessments, Health Phys., 64, 23-36.

Lijinsky, W. and A. Andrews (1980) Mutagenicity of vinyl compounds in Salmonella typhimurium, Teratogen. Car- cinogen. Mutagen., 1, 259-267.

Lyon, M., I.-D. Adler, B. Bridges, L. Ehrenberg, L. Golberg, D. Kilian, S. Kondo, E. Moustacchi, A. Putrament, K. Sankaranarayanan, F. Sobels, R. Sram, G. Streisinger and K. Sundaram (1983) International Commission for Protec- tion against Environmental Mutagens and Carcinogens (ICPEMC), Committee 4 Final Report, Estimation of ge- netic risks and increased incidence of genetic disease due to environmental mutagens, Mutation Res., 115, 255-291.

Mast, R., R. Naismith, R. Sorg, E. Godek, D. Putman and M. Friedman (1983) Mutagenicity studies on acrylamide, Tox- icologist, 3, 38.

Miller, M. and C. McQueen (1986) The effect of acrylamide on hepatocellular DNA repair, Environ. Mutagen., 8, 99- 108.

Miller, M., D. Carter and I.G. Sipes (1982) Pharmacokinetics of acrylamide in Fisher-334 rats, Toxicol. Appl. Pharma- col., 63, 36-44.

Moore, M., A. Amtower, C. Doerr, K. Brock and K. Dearfield (1987) Mutagenicity and clastogenicity of acrylamide in L5178Y mouse lymphoma cells, Environ. Mol. Mutagen., 9, 261-267.

Mukhtar, H., R. Dixit and P. Seth (1981) Reduction in cuta- neous and hepatic glutathione contents, glutathione S-

98 KL. Dearfield et al. /Mutation Research 330 (1995) 71-99

transferase and aryl hydrocarbon hydroxylase activities following topical application of acrylamide to mouse, Toxi- col. I.&t., 9. 153-156.

Neuhauser-Klaus, A. and W. Schmahl (1989) Mutagenic and teratogenic effects of acrylamide in the mammalian spot test, Mutation Res., 226, 157-162.

Oakberg, E. (1968) Mammalian gametogenesis and species comparisons in radiation response of the gonads, in: Ef- fects of Radiation on Meiotic Systems, International Atomic Energy Agency, Vienna, pp. 153-181.

OSHA (Occupational Safety and Health Administration) (1989) Air contaminants; Final rule, 29 CFR Part 1910, Fed. Reg., 54, 2674-2676.

OSHA (Occupational Safety and Health Administration) (1993) 29 CFR Part 1910 - Occupational Safety and Health Standards, Subpart Z - Toxic and Hazardous Substances, Section 1910.1000 Air contaminants.

Pacchierotti, F., C. Tiverson, M. D’Archivio, B. Bassani, E. Cordelli, G. Leter and M. Spano (1994) Acrylamide-in- duced chromosomal damage in male mouse germ cells detected by cytogenetic analysis of one-cell zygotes, Muta- tion Res., 309, 273-284.

Ramsey, J., J. Young and S. Gorzinski (1984) Acrylamide: toxicodynamics in rats, Unpublished report, Dow Chemi- cal Company, Midland, MI (originally reviewed in Dearfield et al., 1988).

Rhomberg, L., V. Dellarco, C. Siegel-Scott, K. Dearfield and D. Jacobson-Kram (1990) A quantitative estimation of the genetic risk associated with the induction of heritable translocations at low-dose exposure: ethylene oxide as an example, Environ. Mol. Mutagen., 16, 104-125.

Russell, L., P. Hunsicker, N. Cacheiro and W. Generoso (1991) Induction of specific-locus mutations in male germ cells of the mouse by acrylamide monomer, Mutation Res., 262, 101-107.

Russo, A., G. Gabbani and B. Simoncini (1994) Weak geno- toxicity of acrylamide on premeiotic and somatic cells of the mouse, Mutation Res., 309, 263-272.

Sakamoto, J. and K. Hashimoto (19861 Reproductive toxicity of a&amide and related compounds in mice - effects on fertility and sperm morphology, Arch. Toxicol., 59. 201- 205.

Sankaranarayanan, K. (1982) Genetic Effects of Ionizing Ra- diation in Multicellular Eukaryotes and the Evaluation of Genetic Radiation Hazards in Man, Elsevier, Amsterdam, 385 pp.

Sega, G. and E. Generoso (1990) Measurement of DNA breakage in specific germ-cell stages of male mice exposed to acrylamide, using an alkaline-elution procedure, Muta- tion Res., 242, 79-87.

Sega, G., R. Valdivia Alcota, C. Tancongco and P. Brimer (1989) Acrylamide binding to the DNA and protamine of spermiogenic stages in the mouse and its relationship to genetic damage, Mutation Res., 216, 221-230.

Sega. G., E. Generoso and P. Brimer (1990) Acrylamide exposure induces a delayed unscheduled DNA synthesis in germ cells of male mice that is correlated with the tempo-

ral pattern of adduct formation in testis DNA, Environ. Mol. Mutagen., 16, 137-142.

Segerbkk, D., E. Faustman, L. Costa, C. Callernan and J. Schroeder, Formation of N-7-(2-carbamoyl-2-hydroxy- ethyljguanine in DNA of the mouse and the rat following intraperitoneal administration of [t4C]-a&amide, Cancer Res., in press.

Shelby, M., K. Cain, L. Hughes, P. Braden and W. Generoso (1986) Dominant lethal effects of acrylamide in male mice, Mutation Res., 173, 35-40.

Shelby, M., K. Cain, C. Cornett and W. Generoso (1987) Acrylamide: induction of heritable translocations in male mice, Environ. Mol. Mutagen., 9, 363-368.

Shiraishi, Y. (1978) Chromosome aberrations induced by monomeric acrylamide in bone marrow and germ cells of mice, Mutation Res., 57, 313-324.

Sobels, F. (1982) The parallelogram: an indirect approach for the assessment of genetic risks from chemical mutagens, in: K. Bora, G. Douglas and E. Nestmann (Eds.), Progress in Mutation Res., 3, 323-327.

Sobels, F. (1989) Models and assumptions underlying genetic risk assessment, Mutation Res., 212, 77-89.

Sofuni, T., M. Hayashi, A. Matsuoka and M. Sawada (1985) Mutagenicity tests on organic chemical contaminants in city water and related compounds. II. Chromosome aber- ration tests in cultured mammalian cells, Eisei Shiken. Hok., 103, 64-75.

Solomon, J., J. Fedyk, F. Mukai and A. Segal (1985) Direct alkylation of 2’-deoxynucleosides and DNA following in vitro reaction with acrylamide, Cancer Res., 45,3465-3470.

Sumner, S., J. MacNeela and T. Fennel1 (1992) Characteriza- tion and quantitation of urinary metabolites of [1,2,3-13C] actylamide in rats and mice using 13C nuclear magnetic resonance spectroscopy, Chem. Res. Toxicol., 5, 81-89.

Sun, L. and B. Singer (1975) The specificity of different classes of ethylating agents toward various sites of HeLa cell DNA in vitro and in vivo, Biochemistry, 14, 1795-1802.

Tanii, H. and K. Hashimoto (1981) Studies on in vitro metabolism of acrylamide and related compounds, Arch. Toxicol., 48, 157-166.

Tripathy, N., K. Patnaik and M. Nabi (1991) Acrylamide is genotoxic to the somatic and germ cells of Drosophila melanogaster, Mutation Res., 259, 21-27.

Tsuda, H., C. Shimizu, M. Taketomi, M. Hasegawa, A. Hamada, K. Kawata and N. Inui (1993) Acrylamide; induc- tion of DNA damage, chromosomal aberrations and cell transformation without gene mutations, Mutagenesis, 8, 23-29.

UNSCEAR (United Nations Scientific Committee on the Effects of Atomic Radiation) (1986) Genetic and Somatic Effects of Ionizing Radiation, Report to the General As- sembly, United Nations, New York.

USEPA (U.S. Environmental Protection Agency) (1987) As- sessment of airborne exposure and dermal contact to acrylamide during chemical grouting operations, Office of Toxic Substances, USEPA Publication No. EPA 560/5-87- 009.

K L. Dearfield et al. /Mutation Research 330 (1995) 71-99 99

USEPA (U.S. Environmental Protection Agency) (19911 40 CFR Parts 141, 142, and 143 National Primary Drinking Water Regulations; Final Rule, Fed. Reg., 56, 3558.

USEPA (U.S. Environmental Protection Agency) (1994) Hu- man genetic risks from exposure to chemicals, focusing on the feasibility of a parallelogram approach, Proceedings of the EC/US Workshop on Risk Assessment October 11-14, 1993, EC Publication No. EUR 15606 EN, USEPA Publi- cation No. EPA/600/R-94/042.

USFDA (U.S. Food and Drug Administration) (1994) Food and Drugs, U.S. Code of Federal Regulations, Title 21 (21 CFR 173.5; 173.10; 173.3101.

Valdivia, R., N. Lafuente and M. Katoh (1989) Acrylamide-in- duced chromosome-type aberrations in spermiogenic stages evaluated in the first cleavage metaphases in the mouse, Environ. Mol. Mutagen., 14 (Suppl. 151, 205.

Voogd, C., J.J. van der Stel and J.J.J.A.A. Jacobs (1981) The mutagenic action of aliphatic epoxides, Mutation Res., 89, 269-282.

Warr, T., J. Parry, R. Callander and J. Ashby (1990) Methyl

vinyl sulphone: a new class of Michael-type genotoxin, Mutation Res., 245, 191-199.

Waters, M. and C. Nolan (19941 Meeting report of the EC/US workshop on genetic risk assessment: ‘Human genetic risks from exposure to chemicals, focusing on the feasibil- ity of a parallelogram approach, Mutation Res., 307, 411-424.

Working, P., K. Bentley, M. Hurtt and K. Mohr (1987) Com- parison of the dominant lethal effects of acrylonitrile and acrylamide in male Fischer 344 rats, Mutagenesis, 2, 215- 220.

Xiao, Y. and A. Tates (19941 Increased frequencies of mi- cronuclei in early spermatids of rats following exposure of young primary spermatocytes to acrylamide, Mutation Res., 309, 245-254.

Zeiger, E., B. Anderson, S. Haworth, T. Lawlor, K. Mortel- mans and W. Speck (19871 Salmonella mutagenicity tests. III. Results from the testing of 255 chemicals, Environ. Mutagen., 9 (Suppl. 91, l-110.