Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

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

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restriction
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
one test with DMSO
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The tk+/tk-, 3.7.2C heterozygote of L5178Y mouse lymphoma cells was obtained from Dr. D. Clive, Burroughs Wellcome Co., Research Triangle Park, NC 27709, and stored in liquid nitrogen.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
10% Post-mitochondrial supernatant fractions of liver homogenates (rats aroclor 1254)
Test concentrations with justification for top dose:
0, 225, 450, 900, 1800, 3600 µg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 3-methylcholanthrene (with S9) and methyl-methanesulphonate (without S9)
Details on test system and experimental conditions:
Each experiment, other than the initial toxicity test, normally consisted of the following groups: vehicle control, four cultures; positive control, two cultures; at least five test compound concentrations, two cultures per concentration. With any chemical, the first experiment was a toxicity test in which cell population expansion was measured. Ten-fold differences in test compound concentrations were used in the toxicity test, the highest being 5 mg/ml unless a much lower concentration was indicated by the poor solubility of a compound.
Evaluation criteria:
See tables 1 and 2
Statistics:
yes
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Insolubility, rather than toxicity, restricted dosing to 3600 µg/ml, which, in the absence of S9 mix, failed to induce a mutagenic response in two experiments. In the presence of S9 mix statistically significant increases in mutant fraction occurred in two experiments. The LOED was 1800 µg/ml, where toxicity was either slight or not demonstrable. See tables 3-6.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 3 : Results of ETU without S9 (trial no.1)

Concentration µg/ml

Cloning efficiency (%)

Relative total growth (%)

Mutant colony count

Mutant fractiona

Group average mutation fraction

DMSO

99

103

107

51

-

0

86

96

81

32

37

86

102

78

30

85

100

79

52

225

102

108

99

33

33

92

99

93

34

450

94

107

58

21

28

95

96

101

36

900

73

85

72

33

-

1800

71

67

86

40

46

63

80

97

51

3600

64

74

85

44

36

92

89

76

28

MMSb

35

24

132

126

154*

36

21

196

181

aMutation colonies per 10^6 clonable cells

bpositive control, Methymethanesulfonate, 2 assays, 15 µg/ml

* P<0.05

Table 4 : Results of ETU without S9 (trial no.2)

Concentration µg/ml

Cloning efficiency (%)

Relative total growth (%)

Mutant colony count

Mutant fractiona

Group average mutation fraction

DMSO

79

99

69

29

-

0

69

93

65

31

26

69

92

40

19

80

116

54

21

225

90

104

71

26

21

83

111

38

15

450

82

109

48

20

19

90

1014

49

18

900

102

96

55

18

17

86

102

44

17

1800

86

96

41

16

15

90

98

36

15

3600

78

98

42

18

16

84

104

35

14

MMSb

44

35

172

130

126*

43

37

157

122

aMutation colonies per 10^6 clonable cells

bPositive control, Methymethanesulfonate, 2 assays, 15 µg/ml

* P<0.05

Table 5 : Results of ETU with S9 (trial no.1)

Concentration µg/ml

Cloning efficiency (%)

Relative total growth (%)

Mutant colony count

Mutant fractiona

Group average mutation fraction

DMSO

63

107

28

15

-

0

77

101

46

20

17

61

81

36

20

71

111

27

13

225

61

87

53

29

25

63

97

39

21

450

73

109

35

16

17

65

104

37

19

900

68

68

51

25

26

82

92

66

27

1800

91

118

98

36

35*

73

97

73

35

3600

83

102

81

33

35*

85

98

94

37

MCAb

56

32

223

132

130*

51

32

195

128

aMutation colonies per 10^6 clonable cells

bPositive control, 3-mthylcholanthrene, 2 assays, 2.5 µg/ml

* P<0.05

Table 6 : Results of ETU with S9 (trial no.2)

Concentration µg/ml

Cloning efficiency (%)

Relative total growth (%)

Mutant colony count

Mutant fractiona

Group average mutation fraction

DMSO

69

96

45

22

-

0

86

104

71

28

23

82

106

47

19

72

95

51

23

225

72

95

48

22

25

84

109

72

29

450

87

87

103

40

37*

84

89

88

35

900

75

77

97

43

40*

65

69

73

38

1800

78

71

103

44

46*

73

92

104

47

3600

73

80

124

56

55*

70

68

113

54

MCAb

69

29

490

238

269*

47

25

418

300

aMutation colonies per 10^6 clonable cells

bPositive control, 3-mthylcholanthrene, 2 assays, 2.5 µg/ml

* P<0.05


Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
negative without metabolic activation

Positive result was only observed when using metabolic activation.
Executive summary:

Seventy-two chemicals were tested for their mutagenic potential in the L5178Y tk+/- mouse lymphoma cell forward mutation assay. Cultures were exposed to the chemicals for 4 hr, then cultured for 2 days before plating in soft agar with or without trifluorothymidine (TFT), 3 µg/ml. The chemicals were tested at least twice. Significant responses were obtained with ETU with S9 (negative result without S9).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 473)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Female Chinese hamster lung fibroblasts (CHL/IU)
Details on mammalian cell type (if applicable):
There were at least 25 modes of the chromosomes, and since the chromosomes are relatively large, they have the advantage of easily observing the
samples, and so are widely used in chromosomal aberration studies using cultured cells.
The cells were purchased on March 5, 2002 from Dai Nippon Pharmaceuticals (passage number at purchase: 14), and were sub-divided into 1mL by adding dimethylsulfoxide (hereafter DMSO, Kanto Chemicals (Co.), Lot No. 210G1441) with a maximum proportion of 10 v/v% relative to the cell suspension, and then stored frozen in liquid nitrogen (passage number at freezing: 17). Cells in the same frozen lot were subject to evaluation of characteristics, and confirmation was made of 25 chromosomes, cell cycle of 16.4 hours and negative mycoplasm. For this study, cells from this frozen lot were thawed and cultivated, and used within four weeks after thawing (passage number: 20~24). Incubation of cells was conducted using a plastic plate (diameter of 6 cm or 10 cm; Becton Dickinson and Company), in a CO2 gas incubator (set to 5% CO2, 37°C temperature, humidified, NAPCO model 7300 or 6301C).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 derived from the lungs of 7 week old male SD rats subject to enzyme induction in phenobarbital and 5.6-benzoflavone.
Test concentrations with justification for top dose:
Based on the cell growth inhibition test results, the dose for the chromosomal aberration tests were set to 258, 515, and 1030 µg/mL for all of the treatment conditions.
The dose for the positive control substance was set to 0.1 µg/mL of MMC with the –S9 mix and 0.05 µg/mL of MMC with the 24 hour treatment. 20 µg/mL of BP was used with the +S9 mix. These were all doses with known chromosomal aberration induction.
Vehicle / solvent:
Sodium chloride solution.
Solubility in sodium chloride solution: dissolved in 25 mg/ml. Solubility in water: 20 mg/ml.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Sodium chloride
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C, and Benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (MEM medium)

DURATION
- Preincubation period: three days
- Exposure duration: 6 or 24 hours

After rinsing the cell surfaces with Ca2+, Mg2+ free phosphate buffered saline (hereafter, PBS(-)), they were fixed for 10 minutes using methanol, and
after dyeing for 10 minutes in a 3 v/v% Giemsa solution, they were lightly rinsed with water and then dried. The cell growth rate for each of the dyed plates was measured using a cell densitometer

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: one plate per dose

NUMBER OF CELLS EVALUATED: 100 cells for each plate, specifically , 200 metaphase cells per dose.
Observations on structural aberrations were conducted according to the following classifications.
Cells with 25±2 or more than 35 centromeres were excluded.
Chromatid breaks, Chromatid exchanges, Chromosome breaks, Chromosome exchanges (dicentric, cyclic chromosomes and such), Fragments
Gaps were those where the non stained portion of the chromatid was narrower than the width of the chromatid. Other aberrations were classified and recorded but were not included as structural aberrations. With numerical aberrations, those with more than 35 centromeres were counted as polyploidal cells containing endopolyploidy cells.

DETERMINATION OF CYTOTOXICITY : Calculation of the 50% cell growth inhibition dose
For each treatment condition, a survival curve was created using the negative control values as 100%. With all of the treatment conditions, since the cell growth inhibition rate was greater than 50%, calculation of the 50% cell growth inhibition dose (IC50) could not be calculated for the test substance.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Structural aberrations were deemed to be cells with at least one structural chromosomal aberration.
Determination of chromosomal aberration induction by the test substance was conducted under each of the test conditions, with a frequency of appearance of cells with structural aberrations or numerical aberrations of less than 5% deemed to be negative (-), and if one or both were greater than 5% but less than 10%, this was deemed probably positive (±), and if one or both were greater than 10%, this was deemed to be positive (+).
Statistics:
Assays were not performed using statistical method.
Species / strain:
other: Female Chinese hamster lung fibroblasts (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The results of the cell growth inhibition study confirmed that the test substance did not have greater than 50% cell growth inhibition for any of the treatment conditions with and without the S9 mix and with 24 hour treatment.
Based on these results, 258, 515 and 1030 µg/mL were set for all of the treatment conditions in the chromosomal aberration study.
Since the results of the preliminary microscopic evaluation in the chromosomal aberration study indicated 50 or more metaphase cells per plate for all doses and all treatment conditions, samples of all of the plates were targets for observation.
The results of the observations on the specimens in the chromosomal aberration tests showed that the frequency of appearance of cells with structural or numerical chromosomal aberration was 5% or less.
Furthermore, the frequency of appearance of cells in the negative control group with structural or numerical chromosomal aberration was 5% or less
On the other hand, the frequency of appearance of cells in the positive control group with structural chromosomal aberration was 10% or more.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that there was no chromosomal aberration induced by 2 -Imidazolidinethione in CHL/IU cells under these test conditions.
The frequency of appearance of cells with structural or numerical chromosomal aberration was 5% or less for any of the doses and all treatment conditions.
Executive summary:

In vitro chromosomal aberration tests were conducted using 2-Imidazolidinethione on CHL/IU cell strains derived from female Chinese hamster lungs.

Based on the results of the preliminary studies, the doses for the cell growth inhibition tests were 64.4, 129, 258 and 1030 µg/mL for the metabolic activation method (-S9 mix) and metabolic activation method (+S9 mix) during short term treatment, as well as the 24 hour continuous treatment (24 hour treatment) method.

The results did not confirm inhibition of cell growth greater than 50% for the test substance in any of the treatment conditions. Based on these results, the chromosomal aberration tests were set to 258, 515 and 1030 µg/mL for all treatment conditions.

In the results of the observation of specimens in the chromosomal aberration study, the frequency of occurrence of cells with structural aberrations and numerical aberration was less than 5% for all of the treatment conditions and doses.

Therefore, it was concluded that there was no chromosomal aberration induced by 2 -Imidazolidinethione in CHL/IU cells under these test conditions.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Strain
Principles of method if other than guideline:
5 strains (S.typhimurium): TA1535, TA1537, TA1538, TA98, TA100
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: TA1535, TA1537, TA1538, TA98 and TA100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
arochlor-induced liver microsomal fraction (10%)
Test concentrations with justification for top dose:
5, 50, 500, and 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
The mutation test was performed using the procedure described by Ames et al. (1975). Each compound was tested initially at four concentrations with serial tenfold dilutions being made from the top concentration. Triplicate plates were used at each dose.
Evaluation criteria:
A positive response is claimed of the increase in the number of revertant colonies is at least 2-2.5 times the control with some evidence of a dose response relationship.
Statistics:
no data
Species / strain:
other: TA1535, TA1537, TA1538, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
at all concentrations
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The highest dose level used in the mutation study was that concentration which caused slight toxicity.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table of results: Mean number revertant colonies per plate

ETU dose (µg/plate)

TA1535

TA1537

TA1538

TA98

TA100

S9

-

+

-

+

-

+

-

+

-

+

5000

8

12

3

5

8

14

50

50

55

27

500

8

11

5

4

9

12

50

59

52

27

50

8

8

5

8

5

9

54

67

37

22

5

8

12

6

5

6

12

47

58

36

21

Solvent (DMSO)

9

12

5

6

10

11

57

62

39

21

Conclusions:
Interpretation of results (migrated information):
negative

Negative result of ETU mutagenicity.
Executive summary:

Forty-two coded compounds were submitted for testing in the Ames Salmonella/Microsome assay using S. typhimurium strainsTA1535, TA1537, TA1538, TA98,and TA100.The test was performed in the presence and absence of metabolic activation provided by a supplemented Aroclor-induced Iiver microsomal fraction (S9 mix). ETU gave negative results at all concentrations in all strains.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

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 for 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 ¿ 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 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.