Ethylene thiourea (ETU). A review of the genetic toxicity studies

ELSEVIER Mutation Research 317 (1994) 111-132

m

Genetic Toxicology

Ethylene thiourea (ETU). A review of the genetic toxicity studies

Kerry L. Dear f ie ld *

U.S. Environmental Protection Agency, Office of Pesticide Programs, Health Effects Division (7509C), 401 M St. S. W, Washington, DC 20460, USA

(Received 15 June 1993) (Accepted 26 October 1993)

Abstract

Ethylene thiourea (ETU) is a common contaminant, metabolite and degradation product of the fungicide class of ethylene bisdithiocarbamates (EBDCs); as such, they present possible exposure and toxicological concerns to exposed individuals. ETU has been assayed in many different tests to assess genotoxicity activity. While a great number of negative results are found in the data base, there is evidence that demonstrates ETU is capable of inducing genotoxic endpoints. These include responses for gene mutations (e.g. Salmonella), structural chromosomal alterations (e.g. aberrations in cultured mammalian cells as well as a dominant lethal assay) and other genotoxic effects (e.g. bacterial rec assay and several yeast assays).

It is important to consider the magnitude of the positive responses as well as the concentrations/doses used when assessing the genotoxicity of ETU. While ETU induces a variety of genotoxic endpoints, it does not appear to be a potent genotoxic agent. For example, it is a weak bacterial mutagen in the Salmonella assay without activation in strain TA1535 at concentrations generally above 1000/zg/plate. Weak genotoxic activity of this sort is usually observed in most of the assays with positive results. Since ETU does not appear very potent and is not extremely toxic to test cells and organisms, it is not surprising to find that ETU does not produce consistent effects in many of the assays reviewed. Consequently, in many instances, mixed results for the same assay type are reported by different investigators, but as reviewed herein, these results may be dependent upon the test conditions in each individual laboratory. A primary shortcoming with many of the reported negative results is that the concentrations or doses used are not high enough for an adequate test for ETU activity. There are also problems with many of the negative assays generally in protocol or reporting, particularly with the in vivo studies (e.g. inappropriate sample number a n d / o r sampling times; inadequate top dose employed).

Overall, while ETU does not appear to be a potent genotoxic agent, it is capable of producing genotoxic effects (e.g. gene mutations, structural chromosomal aberrations). This provides a basis for weak genotoxic activity by ETU. Furthermore, based on a suggestive dominant lethal positive result, there may be a concern for heritable effects. Due to the many problems with the conduct and assessment of the in vivo assays, it is worth repeating in vivo

* Corresponding author, Tel.703 305 6780; Fax 703 305 5147. This manuscript has been reviewed by the Office of Preven- tion, Pesticides and Toxic Substances, U.S. Environmental Protection Agency and approved for publication. Approval

does not signify that the contents necessarily reflect the views or policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommenda- tion for use.

0165-1110/94/$26.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1 1 10(93)E0018-S

112 K.L. Dearfield /Mutation Research 317 (1994) 111-132

cytogenetic assays and a dominant lethal assay (with acceptable test procedures and data generation) to determine if these results would continue to support a heritable mutagenicity concern.

Key words: Ethylene thiourea; Genetic toxicity; Review

Contents

A b s t r a c t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

1. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

2. A s s a y e v a l u a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

(A) B a c t e r i a l a ssays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

(B) Y e a s t assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

(C) D r o s o p h i l a assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

(D) M a m m a l i a n cells in c u l t u r e - - g e n e m u t a t i o n assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

(E) M a m m a l i a n cel ls in c u l t u r e - - a b e r r a t i o n assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

(F) In vivo c y t o g e n e t i c s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

( G ) D o m i n a n t l e tha l assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

( H ) U n s c h e d u l e d D N A syn thes i s ( U D S ) assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

(I) S i s t e r - c h r o m a t i d e x c h a n g e ( S C E ) assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

(J) Cell t r a n s f o r m a t i o n assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

(K) G e r m cell a ssays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

(L) O t h e r geno tox ic i t y assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

(M) E T U n i t r o s a t i o n p r o d u c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

3. O t h e r c o n s i d e r a t i o n s a n d c o n c l u s i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

4. A c k n o w l e d g e m e n t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

5. R e f e r e n c e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

I. Introduction

Ethylene thiourea (ETU; CAS Registry No. 96-45-7; chemical name 2-imidazolidinethione) is a heterocyclic thionamide (Fig. 1). ETU introduction into the environment occurs mainly through the use of ethylene bisdithiocarbamates (EBDCs) which are a group of fungicides used to control fungal pathogens (NTP, 1992; USEPA, 1992). E TU is a common contaminant, metabolite and

NH

Fig. 1. S t r u c t u r e o f e t h y l e n e t h i o u r e a ( E T U ) .

degradation product of these fungicides (e.g. see Engst and Schnaak, 1974; Jordan and Neal, 1979; Marshall, 1977; Truhaut et al., 1973). The general population is exposed to ETU primarily from food exposed to EBDCs. It is estimated that 8-12 million pounds of EBDCs are used in the United States per year (USEPA, 1992). Thus the use of and potential exposure to ETU may present environmental, dietary and occupational exposure concerns.

Several potential toxicity concerns are associated with exposure to ETU. These include carcinogenicity, developmental toxicity, thyroid toxicity and mutagenicity. The evidence for carcinogenicity demonstrates that ETU increases the incidence of thyroid follicular cell adenomas and adenocarcinomas in both sexes of mice and rats

I( L. DearfieM / Mutation Research 317 (1994) 111 - 132 113

Table 1 Ethylene thiourea genotoxicity assays

Assay Test system Concentration/ Purity Assay result Reference dose a

1. Gene mutation assays 1.A. Bacterial gene mutation assays Salmonella S. typhimurium 10-25 000 ppm, no Not reversion G46 spot test activation used; specified assay in DMSO

Positive Seiler, 1974

S. typhimurium (1) Crystals Not Positive TA1530, TA1531, (2) 20-80 mg/plate; specified; (TA1530) TA1532, TA1964; no activation used; "grade G46 spot test in DMSO purum"

S. typhimurium 10-20000 #g/p la te ; Not Positive TA1535, TA1536, in DMSO? specified; (TA1535 TA1537, TA1538, "complete without G46 purity" activation)

S. typhimurium 1000/~g/plate; > 98% Negative TA1535, TA1537, in DMSO TA1538, TA98, TA100

S. typhimurium 0.2-2000/.tg/plate > 98% Negative TA1535, TA1537, TA1538, TA92, TA98, TA100

S. typhimurium 100 ~g/pla te ; > 98% Positive (TA98 TA1535, TA1537, in DMSO with 3-MC TA98, TA100 activation)

S. typhimurium Not specified; > 98% Positive (TA98 TA98, TA100 in DMSO with activation)

S. typhimurium 100-5000 ~g /p la te > 98% Negative TA1537, TA98, TA100

S. typhimurium Not specified > 98% Negative TA1537, TA98, TA100

S. typhimurium Not specified; > 98% Negative TA1537, TA98, in DMSO TA100

S. typhimurium 5-5000/zg/plate; > 98% Negative TA1535, TA1537, in DMSO TA1538, TA98, TA100

S. typhimurium 0.1-2000/~g/plate; > 98% Negative TA1535, TA1537, in DMSO TA1538, TA98, TA100

S. typhimurium 10-5000/zg/plate; > 98% Positive TA1535, TA1537, in DMSO (TA1535 with TA1538, TA98, & without TA100 activation)

Schupbach and Hummler, 1977

Teramoto et al., 1977; Shirasu et al., 1977

Baker and Bonin, 1981

Brooks and Dean, 1981

Garner et al., 1981

Ichinotsubo et al., 1981b

MacDonald, 1981

Martire et al., 1981

Nagao and Takahashi, 1981

Richold and Jones, 1981

Rowland and Severn, 1981

Simmon and Shepherd, 1981


Table 1 (continued)

Assay

Salmonella reversion assay; fluctuation test

Salmonella reversion assay; Mutascreen ®

Salmonella forward mutation assay

E. coli reversion assay

Test system

S. typhimurium TA1535, TA1537, TA1538, TA98, TA100

S. typhimurium TA98, TA100

S. typhimurium TA1950


S. typhimurium TA1535, TA1537, TA98, TA100

S. typhimurium TA1535, TA1537, TA98, TA100

S. typhimurium TA1535, TA1537, TA98

S. typhimurium TA98, TA100


S. typhimurium TM677

E. coli WP2 hcr

E. coli WP2, WP2 uvrA

E. coli WP2 B/r , WP2 uvrA

E. coli 343/113/uvrB

E. coli WP2 hcr

Concentration/ Purity dose a

4-2500/xg/plate > 98%

0.5-500/zg/plate; > 98% in DMSO

1000-20 000 #g/pla te ; Not no activation specified used; in DMSO

> 500/~g/plate; Not in DMSO? specified

1-100/zg/plate; 98.4% in water

100-10 000/zg / 98.4% plate; in DMSO

10-1000/zg/ml; > 98% in dimethylacetamide

1-500 gg /ml ; > 98% in DMSO

1-1000 brg/ml; Not in DMSO? specified

100 gg /ml ; > 98% in DMSO

10-10 000 p,g/plate Not specified; "complete purity"

0.5-500 gg/pla te ; > 98% in DMSO

Not specified; > 98% in DMSO

200-4000 ~g /ml ; > 98% in phosphate buffer

> 500/zg/plate; Not in DMSO? specified

Assay result

Negative

Negative

Positive

Positive (TA1535 with and without activation)

Negative

Positive (TA1535 with and without activation)

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Positive (galR- & arg + systems with activation)

Negative

Reference

Trueman, 1981

Venitt and Crofton-Sleigh, 1981

Autio et al., 1982

Moriya et al., 1983

Mortelmans et al., 1986 (Case Western Reserve)

Mortelmans et al., 1986 (SRI)

Gatehouse, 1981

Hubbard et al., 1981

Falck et al., 1985

Skopek et al., 1981


Venitt and Crofton-Sieigh, 1981

Matsushima et al., 1981

Mohn et al., 1981

Moriya et al., 1983

KL. Dearfield /Mutation Research 317 (1994) 111-132 115

Table 1 (continued)


E. coli reversion E. coli WP2 uvrA 10-1000 p,g/ml; > 98% Negative Gatehouse, 1981 assay; fluctuation in test dimethylacetamide

E. coli reversion E. coli WP2 uvrA 1-1000 ~g/ml; Not Negative Falck et al., 1985 assay; Mutascreen ® in DMSO? specified

Host-mediated S. typhimurium 500-6000 mg/kg; Not Negative Schupbach and assay G46 in DMSO specified; Hummler, 1977

Swiss albino "grade mouse purum"

Swiss albino S. typhirnurium 670-6000 mg/kg; Not Inconclusive Schupbach and mouse TA1530 in DMSO specified; Hummler, 1977

Male JCL-SD rat S. typhimurium 200-400 mg/kg G46

Male JCL-SD rat

Male JCL-ICR S. typhimurium 200-400 mg/kg mouse G46

Male NMRI mouse S. typhimurium TA1950

1.B. In vitro mammalian gene mutation assays

Mammalian gene Mouse lymphoma mutation assay L5178Y TK ÷/-

Mouse lymphoma L5178Y TK +/-

"grade purum"

Not specified; "complete purity"


Not specified

Chinese hamster ovary (CHO-AT3- 2; several loci)

1.C. In vivo gene mutation assays

Sex-linked recessive D. melanogaster lethal assay

D. melanogaster

1). melanogaster

1). melanogaster

I.D. Yeast and other fungal assays Forward mutation S. pombe

5 mg/kg

140-3000/.~g/ml; in DMSO

225-3600/xg/ml; in DMSO

1000-2000/.~g/ml; in DMSO

0.25-2.5% in sugar water

250 ppm; in DMSO

4900 ppm injection; 12,500 ppm feed; in water

5100 ppm

0.1-1/xg/ml; in DMSO

> 98%

Not specified

> 98%

Not specified

> 98%

97%

98.4%

> 98%

Negative

Negative

Negative

Negative

Inconclusive (with activation)

Negative

Negative

Negative

Inconclusive/ Negative

Inconclusive

Negative



Autio et al., 1982

Jotz and Mitchell, 1981

McGregor et al., 1988

Carver et al., 1981

Mollet, 1975

Valencia and Houtchens, 1981

Woodruff et al., 1985/ Mason et al., 1992

Mason et al., 1992

Loprieno, 1981

116

Table 1 (continued)

Assay Test system

K.L. DearfieM /Mutation Research 317 (1994) 111-132

Concentration/ Purity Assay result dose a

Reference

Reverse mutation

A. nidulans

S. cerevisiae XV185-14C

0.22-116 mM; no activation used; in DMSO

> 98% Negative

88.9-889/zg/ml; > 98% Positive in DMSO (without

activation)

I.E. Plant test Mutation Tradescantia 9.79 x 10 -5 M;

clone 4430 in DMSO?

2. Structural chromosomal alterations

2M. In vitro chromosomal alterations in mammalian cells

In vitro Chinese hamster 1000-3200/xg/ml chromosomal cell line (Don) aberrations

Chinese hamster 1670-5000/zg/ml; ovary (CHO) cells in DMSO

Chinese hamster 6000-10000/zg/ml; ovary (CHO) cells in DMSO

RL I rat liver cell 50-200/zg/ml; no line activation used;

in DMSO

50-400 mg/kg; in aqueous soln

2.B. In vivo chromosomal alterations Bone marrow Male and female cytogenetics Wistar rat

Micronucleus assay Female ICR 150-450 mg/kg; mouse in DMSO

Male and female 700-6000 mg/kg; Swiss albino in gummi arabicum mouse

Male ICR mouse 220-880 mg/kg; in DMSO

B6C3F1 mouse 880-1416 mg/kg; in DMSO

Male and female 220-880 mg/kg; CD-1 mouse in DMSO

Dominant lethal Swiss albino 500-3500 mg/kg; assay mouse in gummi arabicum

Not Positive specified


> 98%

> 98%

> 98%


Not specified

Not specified; "grade purum"

> 98%

> 98%

> 98%

Not specified; "grade purum"

Negative

Positwe(with and without activation)

Negative

Negative

Negative

Negatwe

Negatwe

Negative

Negative

Negative

Positive

Crebelliet a1.,1986

Mehta and von Borstel, 1981

van 't Hofand Schairer, 1982


Natar~an and van Kesteren-van Leeuwen, 1981

NTP, 1992

Dean, 1981


Seiler, 1975


Kirkhart, 1981

Salamone et al., 1981

Tsuchimoto and Matter, 1981


K.L. Dearfield /Mutation Research 317 (1994) 111-132 117

Table 1 (continued).


JCL-ICR mouse 300-600 mg/kg; Not Negative Teramoto et al., 1977; in water with gum specified; Shirasu et al., 1977 arabic "complete

purity"

C3H/HeCr mouse 150 mg/kg; Not Negative Teramoto et al., 1978 in gum arabic soln specified

Reciprocal D. melanogaster 500 ppm; > 98% Negative NTP, 1992 translocations in 5% sucrose soln

3. Other genotoxic effects

3.4. DNA damage a n d / o r repair assays and related tests

Rec assay B. subtilis H17, 20-4000 ~g/disk M45

B. subtilis H17, 2000/zg/disk; M45 spores in DMSO

E. coli WP2, WP67, CM871

E. coli WP2, WP67, CM871

E. coli several deficient strains

S. typhimurium TA1538, TA1978 E. coli K12 and WP2

Inhibition of DNA E. coli polA polymerase I

Lambda prophage E. coli (lysogenic) induction

Not specified

Not specified

500 ~g/ml; in DMSO

125-2000/zg/disk; in DMSO

2273/zg/ml

2-20 mg/ml; only activation used; in DMSO

E. coli SOS E. coli PQ37 Not specified; Chromotest in DMSO

In vitro Human fibroblasts Not specified; unscheduled DNA from skin biopsies in DMSO synthesis (UDS)

HeLa $3 human Not specified; cells in DMSO

WI-38 human fibroblasts

Rat hepatocytes (nuclei isolation)

63-2000/.L g/ml; in DMSO

9.6 × 10-9-3.2 × 10 -3 M


> 98%

> 98%

> 98%

> 98%

Not specified; practical grade

> 98%

> 98%

Not specified

> 98%

> 98%

> 98%

Not specified

Negative Teramoto et al., 1977; Shirasu et al., 1977

Positive Kada, 1981 (without activation)

Negative Green, 1981

Negative Tweats, 1981

Positive (with Ichinotsubo et al., activation) 1981

Negative Rashid and Mumma, 1986

Positive Rosenkranz et al., (without 1981 activation)

Positive Thomsom, 1981

Negative Quillardet et al., 1985

Negative Agrelo and Amos, 1981

Positive Martin and (without McDermid, 1981 activation)

Negative Robinson and Mitchell, 1981

Negative Althaus et al., 1982

118 K L . Dearfield /Mutat ion Research 317 (1994) 111-132

Table 1 (continued)


In vivo/in vitro Female B6C3F1 1500 mg/kg; 98% Negative Frank and Muller, unscheduled DNA mouse hepatocytes in corn oil (UDS); 1988 synthesis (UDS)7'S Positive (S- phase analysis phase increase)

3.B. Sister-chromatid exchange (SCE) assays

In vitro SCE assays Chinese hamster 25-1000/zg/ml; > 98% Negative Evans and ovary (CHO) cells in DMSO Mitchell, 1981

Chinese hamster 1670-5000/xg/ml; > 98% Negative Natarajan and van ovary (CHO) cells in DMSO Kesteren-van

Leeuwen, 1981

Perry and Thomson, 1981

NTP, 1992

Chinese hamster ovary (CHO) cells

Chinese hamster ovary (CHO) cells

In vivo SCE assays Male CBA/J mouse bone marrow and liver

3.C. Yeast and other fungal assays

Mitotic aneuploidy S. cerevisiae D6

Chromosome A. nidulans malsegregation

Mitotic gene S. cerevisiae D4 conversion

S. cerevisiae JD1

Mitotic crossing- over

S. cerevisiae D7

S. cerevisiae T1, T2

A. nidulans

Intrachromosomal recombination

Differential killing

S. cerevisiae RSl12

S. cerevisiae T4, T5

S. cerevisiae 197/2d, rad

0.01-100/zg/ml > 98% Negative

500-10000/xg/ml; > 98% Negative in DMSO

1000 mg/kg; > 98% Negative in DMSO

500 ~g/ml; > 98% Inconclusive in DMSO

19.6-78.3 mM; no > 98% Positive activation used; in DMSO

33-333.33/zg/plate; > 98% Negative in DMSO

50/xg/ml; > 98% Positive in DMSO (without

activation)

2000-4000/zg/ml > 98% Negative

1000/xg/ml; > 98% Negative in DMSO

19.6-78.3 mM; no > 98% Negative activation used; in DMSO

5-40 mg/ml; no Not Positive activation used specified

Not specified; > 98% Negative in DMSO

300-1000 tzg/ml; > 98% Positive (with in DMSO and without

activation)

Paika et al., 1981

Parry and Sharp, 1981

Crebelli et al., 1986

Jagannath et al., 1981

Sharp and Parry, 1981a

Zimmermann and Scheel, 1981

Kassinova et al., 1981

Crebelli et al., 1986

Schiestl et al., 1989

Kassinova et al., 1981

Sharp and Parry, 1981b

K.L. Dear)qeld / Mutation Research 317 (1994) 111-132 119

Table 1 (continued)

Assay Test s y s t e m Concentration/ Purity Assay result Reference dose a

3.D. Cell transformation assays

Cell transformation C3H/10T 1/2 cells

Cell transformation with "promotion"

3.E. Germ cell effects Inhibition of testicular DNA synthesis

Spermhead abnormalities

Syrian hamster embryo (SHE) cells/adenovirus SA7

Syrian hamster embryo (SHE) cells/adenovirus SA7

C3H/10T 1/2 cells

100-1000 p.g/ml; 99.8% in DMSO

62-1000 ~g/ml Not specified

1-24 mM Not specified

Negative McGlynn-Kreft and McCarthy, 1984

Negative Casto, 1975, 1976 in: Heidelberger et al., 1983

Inconclusive Hatch et al., 1986

333 p.g/ml; 99.8% Negative McLeod and in DMSO Doolittle, 1985

Mouse 100 mg/kg Not Negative Seiler, 1977a specified

(CBA X BALB/c) 250-2000 mg/kg > 98% Negative Topham, 1981 F1 mouse in Tween 80

B6C3F1/CRL 166-2655 mg/kg; > 98% N e g a t i v e Wyrobek et al., 1981 mouse in DMSO

a In vitro assays performed with and without exogenous activation unless indicated otherwise or the test system does not normally use such supplementation; solvent is provided if specified in the report.

(Graham et al., 1973, 1975; NTP, 1992; Ulland et al., 1972). ETU also increased the incidence of liver adenomas and carcinomas in both sexes of mice (Innes et al., 1969; NTP, 1992).

ETU has demonstrated developmental toxicity in exposed rodents. A variety of central nervous system and skeletal effects have been induced by ETU exposure to litters of exposed rats (Chernoff et al., 1979; Khera, 1973, 1987). Reduced pup birth weight, litter size and number of viable litters have been observed after exposure to preg- nant mice (Hardin et al., 1987). ETU has been shown to be a developmental toxicant at doses lower than those that produced no apparent ma- ternal toxicity or fetal lethality (Khera, 1973).

Thyroid effects are caused by exposure to ETU (for review, see Hill et al., 1989). Thyroid toxicity includes thyroid hyperplasia, decreased uptake of radioactive iodine by the thyroid and decreased serum levels of triiodothyronine (T3) and te- traiodothyronine (T4). The ability to inhibit iodine localization in the thyroid and the thyroid

toxicity seen may contribute to the thyroid carcinogenic effects induced by ETU (Hill et al., 1989).

ETU has been tested in a wide variety of mutagenicity assays that cover gene mutations, chromosomal alterations and other genotoxic effects. For example, it was included in the Interna- tional Collaborative Program to evaluate short- term tests for carcinogens (de Serres and Ashby, 1981).

The purpose of this review is to examine the available mutagenicity information and data on ETU. The results are tabulated into Table 1. The studies are discussed in the following presenta- tion of the assays.

2. Assay evaluations

(A) Bacterial assays

(1) Salmonel la assay. Many studies have been performed with ETU in the Salmonella assay

120 K.L. Dearfield / Mutation Research 317 (1994) 111-132

with and without metabolic activation. It appears that ETU produces predominantly base substitu- tions as evidenced by positive results in strains TA1535, TA1530, TA1950 and sometimes in strain G46. The EPA's Gene-Tox panel of experts in the Salmonella assay has concluded that ETU is positive in the Salmonella assay based on positive results in strain TA1535 from the Ter- amoto et al. (1977) study (Kier et al., 1986). The addition of metabolic activation does not appear to alter the mutagenic response seen without activation. ETU usually does not induce mutations in the frameshift sensitive strains, e.g. TA1531, TA1532, TA1537, TA1538 and TA1964 with the possible exception of an occasional positive in TA98 (e.g. see several studies as part of the International Collaborative Program, report edited by de Serres and Ashby, 1981; Moriya et al., 1983; Schupbach and Hummler, 1977; Ter- amoto et al., 1977).

Seiler (1974) provided an early indication that ETU was active in the Salmonella assay. ETU at moderate concentrations, 100 and 1000 ppm, produced about a 2.5-fold increase in relative mutagenic activity in a spot test with strain his G46 without activation. This positive result in strain G46 was not confirmed by Teramoto et al. (1977) or Schupbach and Hummler (1977) at concentrations up to 80 m g / p l a t e in plate tests. However, both of these studies were consistent with a base-substitution mechanism as both found mutagenic activity in other base-substitution strains. Schupbach and Hummler (1977) obtained a concentrat ion-dependent increase in the number of revertants induced by ETU, but at fairly high concentrations, 20-80 m g / p l a t e in strain TA 1530. Teramoto et al. (1977) also fQund an increase in revertant frequency in strain TA1535 from 5000 ixg /p la te to 20000 /zg/p la te . Strain TA100 was not responsive to ETU. While strain TA100 was engineered from TA1535, it is well recognized that a differential response between these two strains is not unusual. Further studies by this same laboratory reenforce this finding (e.g. Shirasu et al., 1977). Autio et al. (1982) also found ETU caused a concentrat ion-dependent increase in revertants in strain 1950 at concentrations of 5000 ~ g / p l a t e and above.

Additional studies add to the weight-of-evidence that ETU is mutagenic in Salmonella strain TA1535, usually at concentrations around 1000 izg /p la te and above. Simmon and Shepherd (1981), Moriya et al. (1983) and Mortelmans et al. (1986) all found induced responses of 2-4-fold over background. All other strains tested in these laboratories, including TA1537, TA1538, TA98 and TA100, were negative with and without activation. It is of note that ETU has limited solubility in water and negative results with ETU in this assay were found when dissolved at the limit in water and applied to Salmonella plates at concentrations up to 100 izg /p la te (Mortelmans et al., 1986); ETU is usually dissolved in DMSO for testing (the positive result from Mortelmans et al. was for ETU in DMSO).

ETU was one of the chemicals used in the International Collaborative Program that evaluated short-term tests for carcinogens (de Serres and Ashby, 1981). Several laboratories tested ETU in several Salmonella assays as well as other short-term tests. One positive result, Simmon and Shepherd (1981), mentioned above, showed ETU to produce a concentration related and reproducible increase in TA1535. Two other studies in this program also found positive results with ETU, but with other strains. Garner et al. (1981) found that ETU did not induce revertants in any tested strains (TA98, TA100, TA1535, TA1537) without or with liver postmitochondrial supernatant activation mix from phenobarbital induced rats. However, when activation mix from 3-methyl- cholanthrene induced rats was used, a response was detected in TA98 at 100 ~ g / p l a t e . Ichino- tsubo et al. (1981b) also found ETU to induce revertants over background in TA98 with activation mix from Aroclor 1254 induction. The Pro- gram report did not address these divergences (Bridges et al., 1981).

The studies found in the International Collab- orative Program report appear to support the suggestion that the overwhelming majority of gene mutation studies in Salmonella were negative. However, upon closer examination of the results with ETU in the laboratories that participated in this program, there are a few caveats that would appear to diminish the impact of much of the

I~L. Dearfield /Mutation Research 317 (1994) 111-132 121

negative studies. There were 15 separate report studies involving the use of Salmonella in the Program report (Chapters 21-35 inclusive; de Serres and Ashby, 1981). Three of the reports indicated positive results for ETU (detailed above). Four reports indicated negative results for ETU; however, three of these studies may not have high enough concentrations to detect the weak genotoxic effect of ETU: Baker and Bonin (1981) tested to 1000/zg/p la te ; Brooks and Dean (1981) and Rowland and Severn (1981) tested to 2000/xg/p la te ; Richold and Jones (1981) did test to 5000 /zg/pla te . Four of the studies in the Program used 3 or fewer strains in their evaluations, not including TA1535, which appears to be the strain where ETU most consistently induces mutagenic activity; this restricts the value of the reported negative results for ETU in these studies (Martire et al., 1981; MacDonald, 1981; Na- gao and Takahashi, 1981; Venitt and Crofton- Sleigh, 1981). It should be noted that the Ichinot- subo et al. study mentioned above also did not use strain TA1535. Another study, Trueman (1981) reported that ETU was negative in this Program effort, but ETU was positive in this laboratory when tested on other occasions. Of the remaining three studies, two were not Salmonella plate-incorporation tests but rather fluctuation tests, one using only strains TA98 and TA100 (Hubbard et al., 1981) and one using concentrations up to 1000 / zg /ml (Gatehouse, 1981). The final study was a forward mutation assay using Salmonella strain TM677, a derivation of TA1535 which has the pKM101 R factor plasmid (similar to TA100) (Skopek et al., 1981). This forward mutation assay, which measures resistance to 8- azaguanine, found ETU-negative, but at a highest concentration of 100 /zg/ml . So it appears that the evidence is not overwhelmingly negative, but rather that ETU has weak activity in the Salmonella assay, albeit at concentrations usually above 1000 /xg /p la te usually in strain TA1535.

In a test with an automated assay system (Mutascreen®), ETU was found negative at concentrations up to 1000 / x g / m l in TA1535, TA1537, TA1538, TA98 and TA100 (Falck et al., 1985). This top concentration, again, is at a level where a positive response is usually not seen.

(2) Escherichia coli assays. Several reverse mutation assays with different E. coli strains have been performed with ETU. Teramoto et al. (1977) found ETU to be negative when tested in two strains, WP2 hcr ÷ and WP2 hcr - up to 10000 tzg /p la te with and without activation from phenobarbital induced rat liver. Moriya et al. (1983) and Shirasu et al. (1977) found similar findings in strain WP2 hcr up to 5000 /xg /p la te with and without activation. With the Mutascreen ®, Falck et al. (1985) found a negative result with WP2 uvrA at concentrations up to 1000 / zg /ml with and without activation.

Several studies were performed with E. coli in the International Collaborative Program (de Ser- res and Ashby, 1981). Venitt and Crofton-Sleigh (1981) found ETU-negative in two strains, WP2(P) (pKM101) up to 100/.~g/plate and WP2 uvrA(P) (pKM101) up to 500 /zg/pla te , both with and without activation. Matsushima et al. (1981) performed a preincubation before plating the bacte- ria, but also foundnegat ive results in strains WP2 uvrA ( t rp-) , WP2 uvrA (pKM101) ( t rp - ) and WP2 B / r ( t rp - ) with and without activation; the concentrations used were not specified. Mohn et al. (1981) utilized a liquid suspension assay with strain 3 4 3 / l 1 3 / u v r B at the ga lR- and arg + systems. They obtained positive results with both systems under activated conditions at 0.2-1.0 m g / m l which suggests a base-pair substitution mechanism. Higher concentrations (up to 4.0 m g / m l ) produced values lower than control values. Another study, Gatehouse (1981), utilized a microtiter fluctuation assay with WP2 uvrA with and without activation and found no induced activity up to 1000/zg /ml .

Two additional studies were performed in E. coli in the International Collaborative Program. The preferential ability of ETU to inhibit DNA polymerase I deficient E. coli was determined (Rosenkranz et al., 1981). ETU produced a weak positive result up to 2273 ~ g / m l by preferen- tially inhibiting the pol A~- strain (P3478) over that of the pol A + strain (W3110) without metabolic activation in a liquid suspension assay; this activity was not observed in the presence of activation. The induction of prophage lambda in lysogenic E. coli due to mutagenic action that


induces the "SOS" DNA-repair system was examined by Thomson (1981). ETU induced lambda prophage after bacterial exposure to 1 0 0 0 0 / z g / ml in the presence of metabolic activation.

(3) Host-mediated assays. ETU has been tested in several host-mediated assays, using Salmonella strains inoculated into ETU-exposed rodents. Since ETU appeared to be mutagenic to strain G46 (Seiler, 1974), Teramoto et al. (1977) and Schupbach and Hummler (1977) used this strain in the host-mediated assay. Teramoto et al. (1977) obtained negative results in JCL-SD male rats and JCL-ICR male mice after 3 consecutive doses of 200 or 400 m g / k g given orally at 3-h intervals. Shirasu et al. (1977) found similar results in the ICR male mice at a dose of 150 mg/kg . Schup- bach and Hummler (1977) obtained negative re- suits with G46 in ETU-exposed Swiss albino mice up to 6000 mg/kg . However, at this same dose (which is above their reported LD50 of 5400 mg/kg) , a slight induction of the reversion frequency by a factor of 2.37 was noted when Salmonella strain TA1530 was used as the indicator organism. The reviewers of the host-mediated assay for the EPA's Gene-Tox program evaluated this result and reported that this was evidence for a positive decision in the host-mediated assay (Legator et al., 1982). Autio et al. (1982) report that they found a negative result in this assay with 5 m g / k g ETU in male NMRI mice. However, this dose was lower than the one where ETU induced a slight increase.

(4) Bacterial rec assays. Bacterial rec assays were performed with Salmonella, Bacillus subtilis and E. coli strains. Strains either proficient or deficient in DNA repair are exposed to ETU and if the repair-deficient strain is affected to a greater extent, this suggests that the test compound has DNA-damaging activity. Teramoto et al. (1977) did not find apparent differential activity between B. subtilis strains H17 (rec +) and M45 ( rec - ) after exposure to 4000 ~g /d i sk . Rashid and Mumma (1986) assayed ETU in three bacterial repair assays using Salmonella strains TA1538/ TA1978 (to 2000 /~g/disc), E. coli K-12 strains (polA~-/polA~-) and the E. coli WP2 system

(WP2, WP2 uvrA, WP67, CM611, CM571) (concentrations not provided for E. coli systems). They obtained negative results in these three repair assays.

Several studies were performed in the Interna- tional Collaborative Program. Kada (1981) used spores of H17 and M45 instead of the usual vegetative cells and found a reproducible weak positive result without activation at 2 mg/disk. Green (1981) and Tweats (1981) both used E. coli WP2 (DNA-repair-proficient) and the repair-deficient E. coli strains WP67 uvrA polA and CM871 uvrA recA lexA and obtained negative results with and without activation; exact concentrations used not specified. Ichinotsubo et al. (1981a) used recombinational deficient E. coli strains JC2921, JC9238, JC8471 and JC5519 and 2 strains with suppressor genes, JC7689 and JC7623, either with or without metabolic activation. ETU induced a positive response in all strains with activation but JC7689 at a concentration of 500 p~g/ml.

(B) Yeast assays Several yeast assays were performed under the

auspices of the International Collaborative Pro- gram which used ETU as a test chemical. In a yeast reversion assay via mutagenic action, Mehta and von Borstel (1981) found a greater than 2-fold increase in reversion frequency in Saccharomyces cerevisiae strain XV185-14C after exposure to 88.9 and 889 l zg /ml in stationary phase cells. Negative results were obtained in the presence of metabolic activation and questionable increases were noted when logarithmic growing cells were exposed without activation at 100 Izg/ml. Using Schizosaccharomyces pombe, Loprieno (1981) did not find an increase in mutation frequency in this forward mutation assay measuring five different genetic loci with and without activation up to 1 /~g/ml.

Other laboratories in the Program examined endpoints such as mitotic gene conversion, mitotic aneuploidy, mitotic crossing-over and preferential inhibition of growth in repair-deficient strains. Three laboratories tested for mitotic gene conversion, but with different yeast strains and obtained different results. Jagannath et al. (1981)

K.L. Dearfield / Mutation Research 317 (1994) 111-132 123

tested ETU in S. cerevisiae strain D4 at the ade2 and trp5 loci with a preincubation method with and without activation to 333.33 t zg /p la te and obtained negative results. Zimmermann and Scheel (1981) also obtained negative results in S. cerevisiae strain D7 trp 5 locus with and without activation at 2 mg /m l . These latter authors suggest their testing was performed at insufficient concentrations. Sharp and Parry (1981a) however, using strain JD1 of S. cerevisiae at the his4 po- laron and trp5 locus found a 2-4-fold increase in gene conversion with stationary phase ceils without activation with a minimum effective concentration of 50 ~ g / m l (activation was not required). Parry and Sharp (1981) also examined for mitotic aneuploidy in S. cerevisiae strain D6. They report that ETU produced aneuploidy at 500 /xg /ml without activation after treatment with either ini- tially stationary or logarithmic phase ceils. How- ever, the report of the EPA's Aneuploidy effort suggests that this result is inconclusive since con- firmation of aneuploidy was not performed as genetic events that mimic the aneuploid pheno- type were not considered (Resnick et al., 1986); nonetheless, there appears to be an effect caused by ETU that may be indicative of an aneuploidy event (Parry, 1986). Mitotic crossing-over with S. cerevisiae strains T1 and T2 was examined by Kassinova et al. (1981) and no increase was found with and without activation to 1000/zg/ml . These same authors also examined the preferential inhibition of a repair-deficient strain, T4, versus strain T5, which would indicate DNA damage. No dif- ference between the two strains was found (concentration not indicated). Sharp and Parry (1981b) also examined the preferential inhibition of growth in the repair-deficient rad strain versus the repair-proficient 197/2d strain. They, on the other hand, obtained positive results with and without activation to 1000/zg /ml .

Schiestl et al. (1989) developed an intrachromosomal recombination assay in the yeast S. cerevisiae. ETU induced recombination activity by about 3-fold over solvent control at concentrations of 20 and 40 mg /ml ; the increase and high concentrations do not indicate a potent effect. It was also positive by the same fold increase for interchromosomal recombination at 40 mg /ml .

(C) Drosophila assays ETU has been assayed in Drosophila melano-

gaster as the test organism. The first available report was an abstract from Mollet (1975). With a 2.0% concentration of ETU in 5% sugar water, Drosophila mortality was about 50%, but fertility was not reduced and sterility not induced among survivors. There was no induction of sex-linked recessive lethals although the number of lethal tests per dose (up to 2.5% ETU) was too low to be very sensitive, between 1000 and 2000 tests. Dominant lethal effects were increased slightly although the author reports this was not statistically significant. Furthermore, he suggests that ETU affects XY-bearing sperm differently from sperm lacking sex chromosomes.

Valencia and Houtchens (1981; part of Inter- national Collaborative Program) examined ETU in the Drosophila sex-linked recessive lethal assay. They found negative results after a feeding exposure to 250 ppm and examining a total of 10118 lethal tests. However, these authors suggest that this assay was not tested under optimal conditions under the rigid conditions of the Pro- gram. These results have been confirmed by the National Toxicology Program effort. In a pub- lished study (Woodruff et al., 1985), ETU was reported to be equivocal after feeding exposure and negative after injection exposure. However, these results have now been classified as negative by the NTP upon reexamination of these tests at doses up to 5100 ppm (Mason et al., 1992). The NTP (1992) also reports negative results for reciprocal translocations in Drosophila after a 500 ppm exposure.

(D) Mammalian cells in culture - - gene mutation assays

The NTP has examined the mutagenicity of ETU at the tk locus of mouse lymphoma cells (McGregor et al., 1988). ETU was negative in the assay without activation at concentrations up to 3600 /zg /ml , the apparent limit of solubility in DMSO. However, under activated conditions, just over a 2-fold increase was noted at around 3000- 3 6 0 0 /x g /m l with relative total growth of > 73% observed. This single weak response presents inconclusive evidence for ETU genotoxicity in this

124 I£L. Dearfield /Mutation Research 317 (1994) 111-132

assay system. In contrast, Jotz and Mitchell (1981), as part of the International Collaborative Pro- gram, found negative results with and without activation to concentrations of 3000 / z g / m l with a relative total growth of > 71.7%. This top concentration was where the NTP results began to observe suggestive activity.

Carver et al. (1981; part of International Col- laborative Program) tested E T U in the Chinese hamster ovary (CHO) cell line that was heterozy- gous at aprt and tk loci. Other loci examined were hgprt and ATPase loci. The selection agents used were 8-azaadenine, 5-fluorodeoxyuridine, 6-thioguanine and ouabain. There was not an increased mutant frequency at any loci after exposure to 2000 / z g / m l with and without activation with relative cell survival at 0.86 and above.

(E) Mammal ian cells in culture - - aberration assays

Teramoto et al. (1977) exposed the Chinese hamster cell line, Don, to up to 3200 / xg / m l of E T U for 6, 24 or 48 h. The results for aberrations, hyperploidy and polyploidy were negative. Shirasu et al. (1977) reports the same results from this same laboratory. The EPA-sponsored Aneu- ploidy Commit tee agrees with the hyperploidy and polyploidy conclusions (Galloway and Ivett, 1986). The NTP reports that E T U was negative for inducing aberrations in the C H O cell line at concentrations up to 10000 / z g / m l (NTP, 1992).

Two reports are found from the International Collaborative Program. Natarajan and van Kes- teren-van Leeuwen (1981) report that E T U does not produce a concentrat ion-dependent increase in aberrations in cultured C H O cells treated for 1 h with and without activation. However, aberrations were induced at concentrations of 3.33 and 5.0 m g / m l with and without activation. There was a negative response at 1.67 m g / m l , but large increases at 3.33 were seen, with a larger response with activation (e.g. 24 b r e a k s / 1 0 0 cells versus 0.3 for control with $9). At 5.0 m g / m l , there was still an elevated increase, but less than that seen at 3.33 m g / m l . Although positive, this drop at the high concentration is the basis for the author 's no concentrat ion-dependent increase report. Dean (1981) exposed the rat liver (RL 1) cell

line to ETU. This cell line is reportedly able to possess metabolizing capability, therefore, cultures with $9 were not tested. Negative results were tested to 200 ~ g / m l , but this concentration was significantly lower than that used in other studies where activity was observed. The author states that this result is inconclusive as higher concentrations should have been tested, but were not due to constraints to conserve material provided him.

Information from an abstract of an unavailable report (Pilinskaya, 1982) indicates that ETU (1 m g / m l ) incubated with human lymphocytes for 28 h induced about a 2-fold increase in aberrations over that found in control lymphocytes.

(F) In vivo cytogenetics Teramoto et al. (1977) and Shirasu et al. (1977)

report on in vivo aberration results from male and female Wistar rats exposed to ETU. Rats of 3 or 10 weeks of age were given either a single oral dose of 200 or 400 m g / k g or 2-5 (50 m g / kg x 5, 100 m g / k g X 5, or 400 m g / k g x 2) consecutive oral doses at 24-h intervals. Bone marrow was harvested 24 and 6 h after single and multiple treatments, respectively. These investigators did not find an increased aberration frequency in the 3 or 10 week old animals; however, they suggest that the frequency of numerically aberrant cells (including aneuploid and polyploid cells) was higher, though not statistically significant. Sram (1975) reports that E T U increased the frequency of chromosomal aberrations in mouse bone marrow, but no data or concentrations were available for review.

There are several studies that examined for induced micronuclei in exposed animals. Three of these reported studies all exposed mice to 2 treatments of ETU, given 24 h apart and then harvested for bone marrow 6 h after the second dose. All three of these studies reported negative results: in female ICR mice to 150 m g / k g / d a y (Seiler, 1975), in male and female Swiss albino mice to 6000 m g / k g / d a y (Schupbach and Hummler , 1977) and male and female CD-1 mice to 880 m g / k g / d a y (Tsuchimoto and Matter, 1981; part of International Collaborative Pro- gram). The EPA sponsored Gene-Tox program,

K~L. Dearfield /Mutation Research 317 (1994) 111-132 125

in the review of the micronucleus test suggests this sampling time is inappropriate (Heddle et al., 1983). They state the reason is that enucleation of the erythrocyte takes place about 6 -8 h after the final mitosis; this would not allow an opportunity for micronucleus formation in the PCE. As many chemicals also induce cell-cycle delay, this 6-h interval may be especially inappropriate. There- fore, it is recommended that sampling take place no earlier than 12 h after the second treatment. These three studies then may not be examining for micronuclei in an appropriate manner.

Two studies from the International Collabora- tive Program examined for micronuclei with more appropriate sampling times. Salamone et al. (1981) exposed B6C3F1 hybrid mice (sex not stated) to ETU under 2 protocols. Mice were given two i.p. doses of ETU 24 h apart and then sampled at 48 and 72 h for the 1416 m g / k g / d a y doses and at 24, 72 and 96 h for the 880 m g / k g / day doses. The second protocol involved a single i.p. t reatment with 1328 m g / k g and sampling at 48 h. The 1416 m g / k g dose was reported to represent 80% of the LD50. Under both protocols, the authors concluded the results were negative. A slight increase was seen at the top dose at 48 h in the first protocol, but was not seen in a confirmatory test. Kirkhart (1981) dosed male ICR mice with up to 880 m g / k g one time and then sampled at 30 and 48 h. Negative results were also reported. However, in both of these reports, there was uncertainty as evaluated by the Gene-Tox panel of experts, as these two studies did not examine the recommended minimum number of samples (PCE) for determining an actual effect (Heddle et al., 1983). Also, it was not clear if animals from both sexes were used.

(G) Dominant lethal assays Schupbach and Hummler (1977) exposed Swiss

albino male mice to an oral dose of 500, 1000 or 3500 m g / k g and then mated them to 3 females / m a l e / w e e k for 10 weeks. There were statistically significant increases in number of dead implants per female at several doses in the first 5 weeks after exposure (not found at every week at every dose, so the authors suggest that without consistent response, these results are probably nega-

tive). The Gene-Tox panel of experts for the dominant lethal assay assessed these results as positive (Green et al., 1985); a consistent dose response is not necessary for a positive response in this assay.

Teramoto et al. (1977) and Shirasu et al. (1977) report that JCL-ICR mice were given oral doses of 300 or 600 mg E T U / k g / d a y for 5 consecutive days and then mated for 5 weeks with 1 female per mating period (2-4 per iod /week) . There was a negative dominant lethal effect, but no apparent effect on fertility or toxicity as these doses were at the low end of the dose range used in the Schupbach and Hummler (1977) study that obtained positive results. Teramoto et al. (1978), in another dominant lethal study using C 3 H / H e C r mice exposed to 150 m g / k g for 5 days, obtained similar results to their earlier study (Teramoto et al., 1977). Sram (1975) reports that ETU did not increase the frequency of dominant lethal effects after a single or repeated administration in mice. No details or data from this report were available for review.

(H) Unscheduled DNA synthesis (UDS) assays Three reports from the International Collabo-

rative Program examined UDS after ETU exposure. All three utilized human cultured cells and scintillation counting after DNA extraction to quantitate the level of UDS. After a hydroxyurea pretreatment, Robinson and Mitchell (1981) exposed WI-38 human fibroblasts to ETU for 3 h without activation (63-1000 i zg /ml ) and for 4 h with activation (125-2000 tzg/ml) . They report negative findings, but suggest that ETU was not able to be tested at concentrations capable of inducing cytotoxic effects. Martin and McDermid (1981) exposed HeLa $3 cells, pretreated with hydroxyurea, with and without activation for 2.5 h. They report positive results without activation, negative with activation, but no concentrations or data were provided. Agrelo and Amos (1981) also provided no ETU concentrations for their UDS assay that examined human fibroblasts from skin biopsies, pretreated with hydroxyurea and exposed to ETU for 1 h without activation only. Negative results were reported.


Althaus et al. (1982) performed a rat hepatocyte DNA-repair assay using a nuclei isolation procedure for determination of DNA-repair synthesis. Cultures were treated with ETU (3.2 × 10 -3 M to 9.6 × 10 -9 M) for 18 h. Results obtained were negative after liquid scintillation counting of isolated nuclei.

Frank and Muller (1988) obtained negative results for UDS after an in vivo exposure of 1500 m g / k g ETU in corn oil to female B6C3F1 mice and in vitro culture of hepatocytes. On the other hand, an induction of the number of S-phase cells was found, suggesting an effect on proliferation.

(I) S&ter-chromatid exchange (SCE) assays There are several reports that examined for

SCE in cultured Chinese hamster ovary (CHO) cells. The NTP (1992) reports they obtained negative results for SCE in CHO cells at concentrations up to 10000 /zg /ml . Three studies from the International Collaborative Program also report negative results for SCE in CHO ceils with and without activation. Evans and Mitchell (1981) exposed CHO cells for 21.5 h without activation and 2 h with activation with a total culture time of 24 h. Concentrations used were 2 5 - 4 0 0 / x g / m l without activation and 62.5-1000 / zg /ml with activation. These authors suggest that dosing may not have been high enough and no signs of toxicity were reported. Perry and Thomson (1981) exposed CHO cells to ETU for 1 h with and without activation up to 1 0 0 / z g / m l and obtained negative results. However, these authors also suggested that the restriction in concentration range may fail to identify activity as no toxicity was reported. Natarajan and van Kesteren-van Leeuwen (1981) also obtained negative results after CHO cells were exposed to up to 5.0 m g / m l for 1 h with and without activation (it should be noted that this study saw induced aberrations, see above).

Paika et al. (1981, part of International Collab- orative Program) examined for SCE in male C B A / J mice after an in vivo exposure to ETU. Bone marrow and liver were obtained after a partial hepatectomy and bone marrow was also obtained without a partial hepatectomy per-

formed. Negative results were found under all conditions at doses up to 1000 mg/kg .

(J) Cell transformation assays There were two submitted studies to the

USEPA's Office of Pesticide Programs (OPP) examining for cell transformation effects in C 3 H / 1 0 T 1 / 2 ceils (McGlynn-Kreft and Mc- Carthy, 1984; McLeod and Doolittle, 1985). The transformation frequency was not increased for Type III foci in these cells at concentrations up to 1000 # g / m l . These results were considered acceptable. The second study reported to examine for promotion effects where C 3 H / 1 0 T 1 / 2 cells were pretreated with acetone or MNNG and then exposed to 3 3 3 / x g / m l of ETU. The results were negative, but considered unacceptable as more concentrations are required as promoters fre- quently induce erratic and non-dose related effects.

Hatch et al. (1986) reported a slight enhancement of adenovirus SA7 transformation of hamster embryo cells at a top concentration of 12 mM, but this result was not reproducible in a second laboratory. In this interlaboratory study, therefore, they considered these results for ETU-negative. There were two transformation studies reported in the International Collabora- tive Program (Daniel and Dehnel, 1981; Styles, 1981) that used the BHK21 cell transformation assay. Both studies found positive results for inducing ability of BHK21 cells to form colonies in soft agar medium. However, it is known that this assay is not performed much anymore.

(K) Germ cell assays Seiler (1977a) found that ETU at a 100 m g / k g

i.p. dose did not inhibit testicular DNA synthesis in male mice as detected by uptake of tritiated thymidine. Two studies from the International Collaborative Program assayed ETU for spermhead abnormalities. Wyrobek et al. (1981) dosed B 6 C 3 F 1 / C R L mice once a day i.p. for 5 consecutive days and then sacrificed animals 35 days after the first dose. Sperm were examined and no effects on morphology were seen after dosing up to 2655 m g / k g / d a y . In a similar experiment, Topham (1981) examined ( C B A × BALB/c)F1


mice at doses up to 2000 m g / k g / d a y and found negative results.

(L) Other genotoxicity assays Other genotoxicity assays have been per-

formed with ETU. Crebelli et al. (1986) looked for forward mutations (methionine suppressors), mitotic crossing-over and chromosome malsegregation in Aspergillus nidulans. They found that E T U induced the frequency of mitotic segregation at concentrations to 58.7 mM without apparent effect on gene mutations and mitotic crossing-over. The authors suggest that E T U distur- bance of the mitotic segregation of chromosomes may occur via interaction with non-DNA targets. The SOS Chromotest did not find an induction of the gene sfiA as a consequence of induced D N A damage in the SOS system in E. coli (Quillardet et al., 1985). van ' t Hof and Schairer (1982) found that ETU was effective at a minimum concentration of 9.79 x 10 -s M after a 24-h exposure was able to produce mutations in the plant Trades- cantia, clone 4430. This assay involves detecting a phenotypic change in flower color from blue to pink as detected as sectors in the petals or cells in the stamen hairs.

(M) ETU nitrosation products There are suggestions that ureas can be ni-

trosated with sodium nitrite at appropriate pH's. As many ethylene bisdi thiocarbamate-treated products are cooked and eaten, E T U as a metabolite and a contaminant can be exposed to humans under conditions where nitrosation in the stomach may occur. The following reports examine the potential mutagenic activity by such nitrosation products.

In two studies, Seiler (1975, 1977b) examined the effect a combination of ETU and sodium nitrite would have on inducing Salmonella mutation, micronuclei in mice and aberrations in Chi- nese hamsters. In Salmonella strain G46, E T U by itself was found to be a weak mutagen, but in combination with sodium nitrite (1 mM E T U + 5 mM sodium nitrite), very large increases in mutant frequency were obtained. In the micronucleus assay, performed with sampling times that

may be considered inappropriate to detect weak effects (see above discussion, section F), E T U itself was negative. However, E T U + sodium nitrite given p.o. induced very large increases in micronuclei. It was presumed that nitrosation oc- curred in the stomach, yet genotoxic reaction products were able to reach the bone marrow. E T U (300 m g / k g ) + sodium nitrite (100 m g / k g ) given p.o. also induced large increases in chromosomal aberrations in Chinese hamster bone marrow. An exposure to N-ni t roso-ETU (the nitrosated derivative of ETU) at 100 m g / k g i.p. also induced a large increase in Chinese hamster bone marrow aberrations.

Shirasu et al. (1977) found that under acidic conditions ETU + sodium nitrite added together induced large increases in mutant frequency in Salmonella strains TA1535 (160-fold over background) and TA100 (15-fold) and in E. coli strain WP2 hc r - (30-fold). Also, in a host-mediated assay with Salmonella strain G46 as indicator organism, male ICR mice were exposed p.o. to E T U (150 m g / k g ) + sodium nitrite (50 m g / k g ) or N-ni t roso-ETU (125 m g / k g ) . Large increases in mutant frequency were seen where negative results were seen with E T U alone. A dose response was found if the concentration of sodium nitrite was fixed (50 m g / k g ) and E T U doses were increased (1-25 m g / k g ) .

The interaction of E T U and sodium nitrite was examined in the dominant lethal assay (Teramoto et al., 1978). Male C 3 H / H e C r mice were given test article p.o. once a day for 5 days and then mated for 6 weeks. E T U (300 m g / k g ) + sodium nitrite (100 m g / k g ) killed half the animals; N-ni t roso-ETU at 400 m g / k g was lethal. There was no apparent effect with ETU alone, but E T U (150 m g / k g ) + sodium nitrite (50 m g / kg) reduced the percentage pregnancy, number of implants and number of live embryos at weeks 5 a n d / o r 6. Post-implantation losses were not observed. Similar results were seen with 100 m g / k g N-nitroso-ETU. The authors suggest that interaction of ETU and sodium nitrite is capable of adverse effects in mouse germ cells at pre-meiotic stages.

E T U is metabolized in mice by oxidation of the sulfur atom to 2-imidazolin-2-yl sulfenate.

128 K.L. Dearfield / Mutation Research 317 (1994) 111-132

Autio et al. (1982) tested ETU, this oxidation product and their nitrosated derivatives in the Salmonella assay and the host-mediated assay. E TU by itself was weakly positive and the sulfe- hate was negative in the plate-incorporation assay. However, both of their nitrosated derivatives induced large increases in the Salmonella assay strain TA1950 ( > 5000 revertants per plate) with the ETU derivative more active. In the host- mediated assay with NMRI mice and Salmonella strain TA1950, negative results were obtained with the parent compounds. The nitrosated derivatives produced positive results with the ETU derivative again more active.

It appears from this body of evidence that the nitrosated form of ETU is a mutagenic product and presents a mutagenicity concern.

3. Other considerations and conclusion

Although the available studies on ethylene thiourea (ETU) show many negative results, there is evidence to suggest that ETU has weak genotoxic activity. This is based on responses that are found in several different assay systems (e.g. strain TA1535 in the Salmonella assay, aberrations in cultured mammalian cells, a dominant lethal assay, bacterial rec assay and several yeast assays). The responses are not considered potent and are usually found at higher concentra t ions /doses in the assay systems. Because of the weak activity and potency of ETU, many negative studies may not have been performed with high enough concentrations to adequately test ETU. Higher concentrations in some of these studies might reveal ETU to be weakly positive with additional testing.

This conclusion is consistent with the final position of the U.S. Environmental Protection Agency's (USEPA's) Office of Pesticide Programs (OPP) in its conclusion of the special review of EBDCs (USEPA, 1992). This special review examined the toxicity profile of ETU as well as the EBDCs. These two conclusions are similar because they are derived from the same review effort.

Other groups have made inferences about the ETU genotoxicity data base and while on the surface appear conflicting, they generally do not diametrically disagree with the basic assumption that ETU has some genotoxic activity. During the OPP consideration of EBDCs and ETU, the OPP's Scientific Advisory Panel (SAP; a panel of external peer reviewers) opined " that the evidence for genotoxicity was equivocal both in prokaryotic and eukaryotic test systems" (USEPA, 1992). This opinion was delivered in the context of the carcinogenicity consideration of ETU and was part of the overall evidence for cancer induction. Since the SAP was not examining the genotoxicity information in depth, this opinion recognizes the occurrence of positive and negative results in the data base by their equivocal conclusion. This suggests that this is not a definitive decision and one at least cannot conclude that ETU does not have any genotoxicity activity at all.

The United Kingdom's Ministry of Agricul- ture, Fisheries and Food (MAFF) also examined some of the genotoxicity information on EBDCs and ETU (MAFF, 1990). They concluded that " the mutagenicity s tudies . . . show no evidence that E T U . . . is genotoxic in vivo." Although there are many genotoxicity tests performed with ETU, in actuality, there are very few available in vivo studies in which there were not problems (discussed in assay sections above). Also, one of the dominant lethal studies was judged to have a positive effect (Green et al., 1985). This suggests at the very least that there may be activity for dominant lethal activity. Due to the paucity of well conducted in vivo studies and the suggestive dominant lethal result, it is clear that additional in vivo studies are warranted before a definitive conclusion can be reached on the in vivo activity of ETU.

The International Agency for Research on Cancer (IARC) presented genotoxicity data on ETU (IARC, 1987) in the genetic activity profile methodology as developed by Waters and co- workers (e.g. Garret t et al., 1986). The ETU profile also shows the mixed results of the ETU data base with negative and positive results. In their deliberations on the ETU genotoxicity data


base, I A R C conc luded that the evidence for activity in shor t - te rm tests (i.e. mutagenici ty) is lim- i ted ( IARC, 1982). Limited is def ined a s . . . " w h e n there were at least two positive results, e i ther for different endpo in t s or in systems represen t ing two levels of biological complexity." So it again suggests f rom these de l ibera t ions that E T U is capable of some genotoxic activity.

The Nat ional Toxicology Program also discussed the mutagenic i ty data in a summary form in the Technica l Repor t for the carcinogenici ty studies on E T U (NTP, 1992). The Technica l Re- port also recognizes the many negative studies with some positive results. The test ing pe r fo rmed u n d e r the auspices of the NTP and repor ted in the Technica l Repor t show positive results in the Sa lmonel la assay that are consis tent with other reports. Also, inconclusive activity is seen with the mouse lymphoma assay and negat ive results for Drosophi la tests and aber ra t ions and SCE in C H O cells. Dur ing the del ibera t ions that led to the carcinogenici ty conclusions found in the Technica l Repor t , the genotoxicity evidence was discussed. In the summary minu te s of the NTP ' s Board of Scientific Counse lors ' peer review on drafts of the Technica l Repor t (November 20-21, 1989), the op in ion was p resen ted that " the evidence suppor ted E T U as be ing mutagen ic . " This is not inconsis tent with the overall conclusion

p resen ted in this review. It is clear from these de l ibera t ions and this

review that E T U exhibits weak genotoxic potential. At the very least, it canno t be conc luded that E T U is devoid of any genotoxic activity. In terms of how this may impact on her i table risk and the induc t ion of tumors, such as E T U - i n d u c e d thyroid tumors, is unclear . These are areas of future research that may shed insight on the potent ia l role of E T U in these processes.

4. Acknowledgement

The au thor acknowledges the helpful com- ments and review of this manuscr ip t by Dr. A lb in Kocialski, Office of Pesticide Programs, U.S. En- v i ronmenta l Protec t ion Agency.

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