Carbamic acid, N-[(butylthio)thioxomethyl]-, butyl ester

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-03-31 - 2015-05-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also conducted according to GLP principles

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

Deviation: The positive control EMS induced DNA damage in stomach cells 1.7-fold which was just below the acceptance criterion of 2-fold that was mentioned in the protocol.
Evaluation: The DNA damage expressed as tail intensity of both the vehicle control and positive control are within the historical data control range. Moreover, the positive control induces a statistical significant induction of the tail intensity (%). The acceptance criteria are therefore passed. The fact that this induction was just below two fold (stricter acceptance criteria added by the laboratory) has no effect on the validity of the data. The study integrity was not adversely affected by the deviations.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

476-230-4

EC Name:

-

Cas Number:

39142-36-4

Molecular formula:

C10H19NO3S

IUPAC Name:

n-Butyoxycarbonyl-O-n-butyl thionocarbamate

Test material form:

liquid

Specific details on test material used for the study:

- IUPAC Name: n-Butyoxycarbonyl-O-n-butyl thionocarbamate
- Synonyms: O,O-dibutyl imidothiodicarbonate, S-10036
- CAS number: 39142-36-4
- Form: Liquid
- Lot/batch No.of test material: S20477-148
- Storage condition of test material: At room temperature, in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wil research
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 146 +/- 8.2 g (124-158g)
- Assigned to test groups randomly: yes
- Fasting period before study: limited amount of food overnight the day before dosing
- Housing: group housed (max 5 per cage by sex)
- Diet (e.g. ad libitum): ad libitum pelleted rodent dient (from SSNIFF)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 21
- Humidity (%): 44-92 (due to cleaning procedures, temporary devation occurred, based on historical data, does not affect study integrity)
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Concentration of test material in vehicle:
- Amount of vehicle (if gavage or dermal):

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved/diluted with Arachis oil. The concentrations were dosed within 3 hours after preparation.
Accuracy, homogeneity and stability of the preparations were analysed.

Duration of treatment / exposure:

3days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

80 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

160 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylmethanesulphonate
- Route of administration: oral
- Doses / concentrations: 200 mg/kg

Examinations

Tissues and cell types examined:

Liver and stomach cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Range-finding test - 160 mg/kg was the maximum tolerated dose for three-day repeated exposure.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The first dose was administered at time 0h. The second and third dose were administered ca. 24h and 45h after the first dose.
The positive control was administered at time 24 and 45h.

DETAILS OF SLIDE PREPARATION:
Approximately 3 hours after the third treatment with the test compound or vehicle and second treatement with the positive control, tissues were collected/isolated and examined for DNA damage with the alkaline comet assay.

Liver - A portion of 0.7g from the liver was removed and minced thoroughly on aluminium foil in ice. The minced liver tissue was added to 10 mL of collagenase dissolved in HBSS and incubated in a shaking waterbath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris. The supernatant was collected and centrifuged to precipitate the cells. The supernatant was removed and the cell pellet was resuspended in ice cold HBSS and kept on ice.

Stomach - The stomach was cut open and washed free from food using cold Hank's Balanced salt solution. The stomach was stored on ice in HBSS. It was transferred to a tube containing mincing buffer (EDTA+DMSO+HBSS). It was incubated in this buffer on ice for 15-30 min. After incubation, the fore-stomach was removed and discarded. The surface epithelia of the glandular epithelia was gently scraped one time softly. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days.The stomach was then minced thoroughly on aluminium foil in ice. The minced tissue was added to 10 ml of mincing buffer and incubated in a shaking waterbath at 37 °C for 15 minutes. Thereafter, a low centrifugation force was applied two times to remove undigested debris. The supernatant was collected and filtered through a 100 µm Cell Strainer to purify the cell suspension. The purified cell suspension was centrifuged to precipitate the cells and the cell pellet was resuspended in ice cold HBSS and kept on ice.

METHOD OF ANALYSIS:
The viability of the cells after isolation was determined by manually counting the number of viable cells using trypane blue staining.

20 µL of cell suspension was added to 280 µL of melted low melting point agarose. It was mixed and 50 µL was layered on a precoated Comet slide in duplicate. Three slides per tissue were prepared. The slides were incubated for 15 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area. The cells on the slides were immersed overnight in prechilled lysis solution in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer. The slides were then placed in freshly prepared alkaline solution for 20-33min at room temperature in the dark.
The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 1 Volt/cm. The electrophoresis was performed for 30min under constant cooling. After the slides were rinsed in neutralization buffer. The slides were subsequently immersed for 5 minutes in 70% ethanol and allowed to dry at room teperature. Finally, they were stained for 5 minutes with the fluorescent dye SYBR Gold.

The following criteria for scoring of Comets were used:
* only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
* cells that showed overlap or were not sharp were not scored

Evaluation criteria:

It is acceptable if:
* the percentage tail intensity of the solvent control is within the laboratory historical control data range
* the positive control EMS produces at least a 2-fold statistically significant increase in the percentage tail intensity compared to the vehicle treated animals.

A test compound is considered positive if:
* it induces a biologically as well as a statistically significant dose-dependent increase in percentage tail intensity

Results and discussion

Test results

Sex:

male

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Viability of the cell suspensions: 98-100%

Liver - No statistically significant increase in the mean tail intensity.
Stomach - A dose related statistical significant increase of the tail intensity was observed in stomach cells. The DNA damage expressed as percentage tail intensity increased from 53.18% in the vehicle controls to 64.82%, 69.93% and 72.92% after treatment with 40, 80 and 160 mg/kg, respectively. A Cochran Armitage trend test was not performed given the clear dose response observed.

The positive control tail intensities were within the historical control data range: 92.38%.

Clinical signs: lethargy, ataxia, ventral recumbancy and rough coat at 160 mg/kg; lethargy at 80 mg/kg; no effects at 40 mg/kg and in both controls.

The measured concentrations were in agreement with target concentrations (94-99%). The formulations were homogenous and stable.

Any other information on results incl. tables

LIVER CELLS	Tail intensity (%)	S.D.
Vehicle control	3.24	2.35
Test material 40 mg/kg	3.36	0.86
Test material 80 mg/kg	2.09	0.28
Test material 160 mg/kg	2.62	0.91
Positive control EMS 200 mg/kg	92.71*	3.83
STOMACH CELLS	Tail intensity (%)	S.D.
Vehicle control	53.18	9.61
Test material 40 mg/kg	68.82*	5.7
Test material 80 mg/kg	69.93*	5.44
Test material 160 mg/kg	72.92*	7.19
Positive control EMS 200 mg/kg	92.38*	6.14

* p < 0.05

Applicant's summary and conclusion

Conclusions:

Interpretation of results: positive
The test material does not provoke DNA damage in the Comet assay in liver cells up to a dose of 160 mg/kg but it does cause statistically significant dose related DNA damage in stomach cells.

Executive summary:

In a GLP-compliant OECD TG 489 study, the test material was tested for its capacity to induce DNA damage when admistered orally by gavage to rats.

Five male rats were administered 40, 80 and 160 mg/kg. Animals were killed 48 hours after the first administration and about 3hours after the third and last admnistration, the liver and stomach were extracted and single cell suspensions from the liver and stomach were made followed by Comet slide preparation. The slides were analyzed and the tail intensity (%) was assessed. Concurrent vehicle and positive controls were also tested.

No statistically significant increase in the mean tail intensity was observed in liver cells. However, a statistically significant dose related increase of the tail intensity was observed in stomach cells. The DNA damage expressed as percentage tail intensity increased from 53.18% in the vehicle controls to 64.82%, 69.93% and 72.92% after treatment with 40, 80 and 160 mg/kg, respectively.

The negative and positive controls fell in the range of the historical control data range.

It is concluded that the test is valid and that the test material does not provoke DNA damage in the Comet assay in liver cells up to a dose of 160 mg/kg but it does cause statistically significant dose related DNA damage in stomach cells.