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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
ETU has been tested extensively for genotoxicity in a variety of in vitro and in vivo systems, and the results, with a few exceptions, are negative. Results of bacterial gene mutation studies with several strains of Escherichia Coli and Salmonella typhimurium were negative, except for isolated positive responses reported with S. typhimurium strains TA1535. Results from studies of genetic effects in yeast showed some potential for induction of mitotic aneuploidy, gene conversion and DNA damage. No induction of sex-linked recessive lethal mutations was observed in germ cells of male Drosophila melanogaster treated with ETU by feeding or injection. ETU was tested in mammalian cells in vitro for induction of chromosomal aberrations, sister chromatid exchanges (SCE) and unscheduled DNA synthesis; all results were negative. Positive results were reported in a mouse lymphoma assay for induction of trifluorothymidine resistance in L5178Y cells. In vivo mammalian tests for induction of micronuclei or sister chromatid exchanges in bone marrow cells of mice were negative, as were tests for induction of dominant lethal mutations or sperm abnormalities.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Mar-Jun 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
ETU was administered to groups of mice by gavage at doses of 100, 300, 1000 or 3000 mg/kg for 2 and 12 hour exposure periods in separate studies. Vehicle control and positive control (dimethylnitrosamine) groups were used for each study. Unscheduled DNA synthesis (UDS) was assessed by measuring 3H-thymidine incorporation into hepatocytes using an autoradiographic method.
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:University of Surrey Breeding unit
- Age at study initiation:no data
- Weight at study initiation:11-31g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:no data
- Housing:in group of 6 mice
- Diet (e.g. ad libitum):RM1 (E), ad libitum
- Water (e.g. ad libitum):not presiced, ad libitum
- Acclimation period:no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C):no data
- Humidity (%):no data
- Air changes (per hr):no data
- Photoperiod (hrs dark / hrs light):no data

Route of administration:
oral: gavage
Vehicle:
The test chemical was suspended in 1.0% (w/v) gum tragacanth.
The animals received the dosing solutions at 10 ml/kg gavage.
Details on exposure:
no
Duration of treatment / exposure:
2 hours of exposure : 7.30-9.00hrs
Frequency of treatment:
single administration
Remarks:
Doses / Concentrations:
100, 300, 1000 or 3000 mg/kg bw (corresponding to 10, 30, 100 and 300 mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
5 males /group, the sixth was to allow for unsucceddful cannulations.
Control animals:
yes, concurrent no treatment
Positive control(s):
yes, dimethylnitrosamine (20 mg/kg bw) dissolved in water
Tissues and cell types examined:
Hepatocytes were isolated 2 and 12 hours after exposure, following a lethal dose of phenobarbital.
Details of tissue and slide preparation:
Mice were sacrified by an ip administration of a lethal dose of Sagatal and the hepatocytes isolated by non-circulating collagenase perfusion. The animals were cannulated by the hepatic portal vein or by the vena cava of the first attempt had been unsuccessful. Hepatocytes from five animals were prepared concommitantly. Livers were perfused at 10ml/min with Ca2+-free bicarbonate buffer for 5 mins then with collagenase solution for 5 mins until soft. The five liver samples were removed to separate beakers of PBS'A' and shaken with forceps to release the hepatocytes. The cell suspension was filtered through 125µm pore size nylon mesh, then washed three times by centrifugation at 50 x g for 2 mins and resuspension of the pellet in PBS'A'. The cells were finally resuspended in complete Leibovitz L-15 medium containing 10% foetal calf serum and 100µg/ml kanamycin. The yield and viability (according to exclusion of trypan blue) were determined. The cells were diluted to 3 x 105 viable cells/ml in complete L-15 and 1.0m1 aliquots seeded onto 22mm round thermanox coverslips in 12-well dishes.Triplicate cultures were prepared from each mouse. Culture dishes were incubated at 37°C in air for 2 hours for cells to attach.
Labelling of Cultures : The medium was removed and the cultures were rinsed with serum-free L-15. (Methyl-3H)-Thymidine(10µCi/ml) in serum free L-15 (1ml) was added to each well ahd the dishes incubated at 37°C for 3 to 4 hours. Cultures were then washed twice with L-15 containing 0.25mM thymidine and incubated overnight in thismedium.
Fixation of Cells: Cultures were washed twice with PBS'A' then treated with 2ml of 1% sodium citrate for 10 minutes to swell the nuclei. The culture were fixed using three changes of ethanol:acetic acid (3:1) for 10 minutes each, then washed with four changes of distilled water, air dried and mounted cell surface uppermost on glass slides using DPX. Each slide was labelied with the study number and a unique slide number. The slides were left for at least 17 hours to set.


Evaluation criteria:
The test chemical is considered negative if the net nuclear count is less than 3 at the highest dose in an experiment in which the positive control displays its usual activity.
Statistics:
Mean net nuclear nounts +/-SEM were determined for each of the triplicate slides per animal and the mean +/-SD net nuclear count and percentage of cells in repair for each mouse were then calculated. From these values the mean +/-SD for each dose group was determined. Differences between groups were analysed by student t test. The test material is considered positive if the mean net nuclear grain count of the treated animals is statistically greater than that of controls and equal to or greater than 3 grains per nucleus (the upper limit of control values).
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No signs of distress were observed in any of the treated animals. However hepatotoxicity was apparent from the cultured hepatocytes following treatment with 1000 and 3000mg/kg ethylenethiourea. In the 2 hour exposure study the hepatocytes from only 2 animals in each of these groups were viable at the end of the culture period. Following 12 hours exposure all cultures were non-viable from the top dose group and viable cultures were observed from 2 out of 5 animals of the 1000mg/kg group and 3 out of 4 animals of the 300mg/kg up. It therefore appear that toxicity was dose and time related.
ETU induced UDS in hepatocytes following a 2 hour exposure at toxic doses only. This occurred in both animals treated with 3000 mg/kg ETU for which viable hepatocyte cultures were found but in only one of the animals treated with 1000 mg/kg ETU. Thus the latter result was not found to be significantly different from control.
Following a 12 hour exposure period ETU did not induce UDS in any of the animals from which hepatocytes survived the culture period. The positive control, DMS, gave a positive result expected.

Table 1 : Hepatocyte UDS following ETU treatment of mice for 2 hours

Dose group

n

Net nuclear grain +/- SD

% in repair +/-SDa

Control

4b

-1.54 +/- 0.87

1.7 +/- 2.7

ETU – 100 mg/kg

5

-2.46 +/- 0.36

0.2 +/- 0.3

ETU – 3000 mg/kg

5

-1.96 +/- 1.4

1.8 +/- 2.7

ETU – 1000 mg/kg

2c

1.41 +/- 3.8

24.8 +/- 27.9*

ETU – 3000 mg/kg

2c

7.58 +/- 1.9*

60.1 +/- 7.9**

DMN – 20 mg/kg

4b

6.15 +/- 4.3*

49.0 +/- 28.4*

Mice were dosed by gavage and sacrified 2 hours later.

aPercentage of cells with net nuclear grain counts of 5 or more.

bHepatocytes were not isolated successfully from the remaining animals due to poor perfusion.

cHepatocytes died in culture from remaining animals indicating toxicity.

* Significantly different from control by Student t test p<0.05

** Significantly different from control by Student t test p<0.01

Table 2: Hepatocyte UDS following ETU treatment of mice for 12 hours

Dose group

n

Net nuclear grain +/- SD

% in repair +/-SDa

Control

2b,c

-2.23 +/- 0.21

0.5 +/- 0.24

ETU – 100 mg/kg

4b

-2.23 +/- 0.30

0 +/- 0*

ETU – 3000 mg/kg

3b,c

-2.36 +/- 0.87

0.6 +/- 0.5

ETU – 1000 mg/kg

2c

-2.49 +/- 0.82

0.3 +/- 0.47

ETU – 3000 mg/kg

0c

-

-

DMN – 20 mg/kg

5

8.68 +/- 1.27*

68.6 +/- 11.0*

Mice were dosed by gavage and sacrified 12 hours later.

aPercentage of cells with net nuclear grain counts of 5 or more.

bHepatocytes were not isolated successfully from the remaining animals due to poor perfusion.

cHepatocytes died in culture from remaining animals indicating toxicity.

* Significantly different from control by Student t test p<0.05

Conclusions:
Interpretation of results (migrated information): negative (expert judgement)
ETU induced unscheduled DNA synthesis in mouse hepatocytes 2 hours after oral administration at 3000 mg/kg. no effect was seen after 12 hours at doses up to 1000 mg/kg. ETU at 1000 and 3000 mg/kg was apparently hepatotoxic following both 2 hours and 12 hours exposure as seen by the poor survival of hepatocytes in culture. It is therefore probable that the positive UDS result obtained at the top dose is related to the toxicity and may not be relevant to exposure to man levels encountered in the workplace.
Executive summary:

ETU was administered to groups of mice by gavage at doses of 100, 300, 1000 or 3000 mg/kg fpr 2 and 12 hour exposure periods in separate studies. Vehicule control and positive control (dimethylnitrosamine) groups were used for each study. unscheduled DNA synthesis (UDS) was assessed by measuring 3H-thymidine incorporation into hepatocytes using an autoradiographic method.

ETU at 3000 mg/kg induced unscheduled DNA synthesis in mouse hepatocytes following a 2 hours exposure, however signs of hepatotoxicity were seen at doses of 3000 mg/kg and above. There was no induction of UDS after 12 hours exposure or at non-toxic doses of ETU after 2 hours exposure. It is therefore unlikely that this represents a true genotoxic response.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

There is an extensive data base on the genotoxicity potential of ethylene thiourea. The genotoxicity of ethylene thiourea has been reviewed (IARC, 2001; Dearfield, 1994; Elia et al.,1995; Houeto et al., 1995). The following summary is quoted as in the IARC monographs.

 

Humans

The frequency of sister chromatid exchange was increased in peripheral lymphocytes of pesticide applicators who had presumably been exposed to ethylenethiourea as a metabolite of ethylenebisthiocarbamate fungicides. In the same study, the exposed individuals also had a higher frequency of chromosomal translocations than controls but not of other types of chromosomal damage (Steenland et al., 1997).

 

Experimental systems

(a)DNA damage

Ethylenethiourea did not induce SOS repair in Salmonella typhimurium (van der Lelie et al., 1997)or Escherichia coli (Quillardet et al., 1985). It induced λ phage in Escherichia coli (Thomson, 1981). It was weakly active in the E. coli polA test for differential toxicity only in liquid suspension (Rosenkranz et al., 1981); it caused differential toxicity in one E. coli rec assay (Ichinotsubo et al., 1981) and equivocal results in two assays in Bacillus subtilis rec (Kada, 1981; Teramoto et al., 1977). Ethylenethiourea induced DNA damage in the yeast Saccharomyces cerevisiae (Sharp and Parry, 1981), as measured by differential survival of repair-deficient strains.

 

(b)Mutation and allied effects in vitro

Ethylenethiourea was not mutagenic in S. typhimurium with or without metabolic activation (Richold and Jones, 1981; Franekic et al., 1994; Gatehouse, 1981; Brooks and Dean, 1981), except in a few base-pair substitution or frameshift strains with metabolic activation (Enomoto et al., 2004); Mortelmans, 1986, Simmons and Shepherd, 1981); Moriya et al., 1983; Garner et al., 1981; Venitt and Crofton-Sleigh, 1981) . No mutation was induced in E. coli (Taramoto et al.,1977; Venitt and Crofton-Sleigh, 1981; Moriya et al.,1983; Gatehouse, 1981), except for a weak response in one study (Mohn et al., 1981). In mouse or rat host-mediated assays, no mutations were induced in S. typhimurium G46 (Teramoto et al., 1977; Schüpbach and Hummler, 1977), but a positive response was seen in S. typhimurium TA1530 in mice ( Schüpbach and Hummler, 1977). Ethylenethiourea did not induce forward mutation in Schizosaccharomyces pombe (Loprieno, 1981), but it induced reverse mutation in Saccharomyces cerevisiae (Mehta and von Borstel, 1981; Parry and Sharp, 1981). It induced mitotic gene conversion in one study but not in others, and induced intrachromosomal recombination and aneuploidy in yeast (Crebelli et al., 1986). Ethylenethiourea marginally induced petite mutants in yeast. There is disagreement in the literature with regard to the mutagenicity of ethylenethiourea at the Tk locus in mouse lymphoma L5178Y cells (Jotz and Mitchell, 1981; MacGregor et al., 1988). It was not mutagenic at multiple loci in Chinese hamster ovary cells with or without S9 (Carver et al., 1981). Ethylenethiourea did not induce chromosomal aberrations (Narumi et al., 2004; Teramoto et al., 1977; Dean, 1981) or sister chromatid exchange (Evans and Mitchell, 1981; Natarajan and van Kesteren-van Leeuwen, 1981; Perry and Thomson, 1981) in cultured Chinese hamster cells or a rat liver cell line or micronuclei in Syrian hamster embryo cells (Fritzenschaf et al., 1993). Ethylenethiourea transformed BHK-21 cells in culture (Styles, 1981) and had weak transforming activity on BALB/c-3T3 cells (Matthews et al., 1993 as quoted in IARC 2001).

 

(c)Mutation and allied effects in vivo

DNA damage, as measured in the Comet assay, was induced in liver, kidney, lung and spleen, but not bone-marrow cells of mice given an intraperitoneal injection of ethylenethiourea (Sasaki et al., 1997). Chromosomal aberrations were not induced in rat bone-marrow cells after oral administration (Teramoto et al., 1977) or in female Chenise hamster lungs (Anonymous 2004), and no sister chromatid exchange was induced in mouse bone-marrow cells after intraperitoneal injection (Paika et al., 1981). Micronucleus formation was not induced in mouse blood or bone-marrow cells after intraperitoneal (Seiler, 1973; Kirkhart, 1981; Tsuchimoto and Matter, 1981; Morita et al., 1997) or oral administration (Schüpbach and Hummler, 1977). Ethylenethiourea did not induce dominant lethal mutations (Schüpbach and Hummler, 1977; Teramoto et al., 1977; Shirasu et al., 1976) or sperm abnormalities (Topham, 1981) or inhibit testicular DNA synthesis in male mice (IARC, 2001). In Drosophila melanogaster, sex-linked recessive lethal mutations were not induced (Woodruff et al., 1985; Mason et al., 1992), but somatic recombination was induced at the w/w+ locus in one of two studies (Vogel and Nivard, 1993 and Rodriguez-Arnaiz, 1997 as quoted in IARC, 2001). Micronuclei and chromosomal aberrations were induced by ethylenethiourea in shallot root tips (Franekic et al., 1994 as quoted in IARC 2001).

 

Additional references

 

IARC (International Agency for Research on Cancer) (2001)Vol.: 79, 659-701.

Dearfield, K.L. (1994) Ethylene thiourea (ETU). A review of the genetic toxicity studies.Mutat. Res.,317, 111–132.

Elia, M.C., Arce, G., Hurt, S.S., O’Neill, P.J. & Scribner, H.E. (1995) The genetic toxicology of ethylenethiourea: A case study concerning the evaluation of a chemical’s genotoxic potential.Mutat. Res.,341, 141–149.

Houeto, P., Bindoula, G. & Hoffman, J.R. (1995) Ethylenebisdithiocarbamates and ethylenethiourea: Possible human health hazards.Environ.Health Perspectives,103, 568–573.


Justification for selection of genetic toxicity endpoint
Several studies are necessary to evaluate the genotoxic potential of a substance.

Justification for classification or non-classification

The data available on genotoxicity do not suggest a classification of ethylene thiourea according to the criteria of Regulation (EC) No 1272/2008 and Directive 67/548/EEC.