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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Other name = Ethylenethiourea (ETU)
Description : white powder
Storage conditions : at ambient temperature and prepared immediately before use.
Batch no.T154
purity = 99.6%

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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).

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.