4-mesyl-2-nitrotoluene

Genetic toxicity: in vivo

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:

2012-05-09 to 2013-01-19 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform study without deviations conducted under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-550-0

EC Name:

-

Cas Number:

1671-49-4

Molecular formula:

C8H9NO4S

IUPAC Name:

4-methanesulfonyl-1-methyl-2-nitrobenzene

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Physical state: off-white powder
- Stability under test conditions: not reported
- Storage condition of test material: At room temperature (< 30 °C)

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 to 11 weeks
- Weight at study initiation: males, first experiment: 253.3 g ± 10.9 g (range 228 to 267.6 g); females, first experiment: 159.9 g ± 5.4 g (range 150.5 to 169.9 g); males, second experiment: 241.5 g ± 7.4 g (range 231 to 254.1 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not reported
- Housing: in groups in Makrolon Type III/IV cages with wire mesh top with granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 to > 90 during acclimatization; Relative humidity 45 - 65% during experimental performance
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark, 12 hours light

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

1% carboxymethylcellulose (CMC)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: on the day of the experiment the test item was suspended in 1% CMC. Grinding in a mortar was necessary to formulate the test item. The test substance was administered as suspension in 1% CMC. the administration volume was 15 mL/kg bw.

Duration of treatment / exposure:

Animals received a single dose of substance in vehicle and were observed for up to 16 hours

Frequency of treatment:

Single application

Post exposure period:

Up to 16 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
Males: 400 mg/kg and 800 mg/kg (additional group of 200 mg/kg was included due to high mortality in the 16-hours post-treatment group receiving 800 mg/kg); Females: 250 mg/kg and 500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

Four

Control animals:

yes, concurrent vehicle

Positive control(s):

4 hours preparation interval: N,N'-dimethylhydrazinedihydrochloride (DMH) at 80 mg/kg bw
16 hours preparation interval: 2-acetylaminofluorene (2-AAF)

Examinations

Tissues and cell types examined:

Isolated hepatocytes from the livers of test animals

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: a maximum tolerated dose was determined in two pre-experiments, in which the substance was administered by oral gavage to two male and two female animals under the same conditions that were applied in the main test

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The washed hepatocytes were centrifuged and transferred into Williams medium E supplemented with:
Hepes 2.38 mg/mL
L-Glutamine 0.29 mg/mL
Penicillin 100 units/mL
Insulin 0.50 µg/mL
Streptomycin 0.10 mg/mL
Fetal calf serum (FCS) 100 µL/mL
This complete medium was adjusted to pH 7.6. Three cultures were established from each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (200000 viable cells/mL) were added to 35 mm six-well dishes containing one 25 mm round plastic coverslip per well coated with gelatine.

After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO2 humidified incubator at 37° C the culture medium was discarded. The cell layer was then rinsed once with physiologically buffered saline (PBS) to remove non-adherent cells. Subsequently, 3HTdR (5 µCi/mL, specific activity 70 - 90 Ci/mmol) in 2.0 mL culture medium (Williams medium E (WME), 1 % (v/v) fetal calf serum (FCS)) was added to the cultures. After a labeling time of 4 h the cells were washed twice with WME supplemented with 1 % (v/v) FCS and 0.25 mM unlabeled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3:1 v/v) for 20 minutes each, rinsed with 96 % (v/v) ethanol, and air-dried.


DETAILS OF SLIDE PREPARATION:
The cover slips were mounted on glass slides, cell side upwards and coated with KODAK NTB photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 4°C . The photographic emulsion was then developed at room temperature, fixed in Fixer and stained with hematoxylin/eosin.

METHOD OF ANALYSIS:
Evaluation was performed microscopically on coded slides using microscopes with oil immersion objectives. Slides were examined to ensure sufficient cells of normal morphology were present before analysis. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the Sorcerer UDS device. In addition, the number of grains of the most heavily labeled nuclear-sized cytoplasm area adjacent to the nucleus was counted. Two slides per animal and 50 cells per slide were evaluated (except for the positive control group of the females with a total of 151 evaluated cells per animal on three slides). Heavily radio-labeled cells undergoing replicative DNA synthesis were excluded from counting.

All animals per group were evaluated as described above, except for the mid dose group of the 4 h preparation interval in male rats and the high dose group of the 16 h preparation interval in female rats where three male animals instead of four animals were evaluated, due to technical reasons during the liver perfusion.

DATA RECORDING
The data generated were recorded in the raw data. The results were presented in tabular form, including experimental groups with the test item, vehicle and positive controls.

The nuclear and cytoplasmic grain counts, the net grain counts (nuclear minus cytoplasmic grains) as well as the mean and percentage of cells in repair (cells with a net grain count larger than 5) is reported separately. Individual slide and animal data are provided. The mean counts with standard deviation are used to describe the distribution of 3HTdR incorporation in the nucleus, the cytoplasm and for the net grains, respectively.

Evaluation criteria:

Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.

A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.

A group average between 0 and 5 net grains is considered as a marginal positive response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains.

Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test.

A test item producing net grains not greater than 0 or not significantly greater than the concurrent control, at any of the test points is considered negative in this system.

Statistics:

A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding vehicle controls.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: first pre-test with 1250 mg/kg in males and 800 mg/kg in females; a second pre-test was conducted due to severe clinical signs occurring in the first pre-test and doses of 800 mg/kg and 500 mg/kg were administered to two male and two female rats, respectively
- Solubility: the substance was suspended in the vehicle
- Clinical signs of toxicity in test animals: first pre-test caused severe clinical signs in animals and two males were sacrificed after 2 to 4 hours and the females were sacrificed moribund after 6 hours
- Rationale for exposure: the oral route was used as this is of relevance to human risk assessment

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: Severe toxicity was noted in the male 800 mg/kg b.w. 16 h treatment group. On this basis additional male treatment groups were dosed at 200 mg/kg b.w., for both time points. Associated positive and negative control groups were also used. Hence, male rats receiving a single oral dose of the substance at dose levels of 200, 400 and 800 mg/kg showed a number of clinical symptoms including reduction of spontaneous activity, abdominal position, ruffled fur, tumbling, apathy, sunken flanks and death (800 mg/kg 16 h only). Female rats receiving a single oral dose of the substance at dose levels of 250 and 500 mg/kg showed a number of clinical symptoms including reduction of spontaneous activity, ruffled fur, sunken flanks, abdominal posture, eyelid closure, tumbling, lacrimation and gasping. the toxic effects seen indicated systemic exposure to the test substance. The animals in the negative and positive control groups did not show any signs of toxicity.
- Statistical evaluation: no statistical evaluation of the results was necessary.

Any other information on results incl. tables

Group means of nucleus, cytoplasmic area and net grains of males

	Nuclear Grain Count		Cytoplasmic Grain Count		Net Grain Counts		Nuclear Grain Counts of Cells in Repair		Cells in Repair
Test Group	Mean	SD	Mean	SD	Mean	SD	Mean	SD	%
	4 h Preparation Interval
Vehicle control (CMC 1 %) 1st experiment	22.58	11.28	38.85	14.61	-16.27	11.97	7.67	1.62	5
Vehicle control (CMC 1 %) 2nd experiment	15.31	7.08	30.00	10.63	-14.69	9.78	2.88	0.88	1
200 mg/kg b.w. test substance 2nd experiment	20.08	7.94	32.89	12.13	-12.81	9.93	5.40	1.23	3
400 mg/kg b.w. test substance 1st experiment*	19.29	6.99	37.54	12.67	-18.25	11.95	8.83	1.18	2
800 mg/kg b.w. test substance 1st experiment	17.07	6.60	32.62	12.14	-15.55	11.18	6.06	0.58	3
positive control (DMH) 1st experiment	55.90	19.65	28.01	11.64	27.88	16.88	30.05	15.42	92
positive control (DMH) 2nd experiment	72.82	21.60	32.17	11.84	40.65	18.40	42.19	16.97	96
	16 h Preparation Interval
Vehicle control (CMC 1 %) 1st experiment	29.33	11.54	44.45	15.66	-15.13	14.34	14.46	9.76	7
Vehicle control (CMC 1 %) 2nd experiment	24.32	10.45	41.27	14.73	-16.95	13.76	11.06	7.06	8
200 mg/kg b.w. test substance 2nd experiment	24.14	9.12	33.31	11.09	-9.17	10.65	10.08	2.95	8
400 mg/kg b.w. test substance 1st experiment	29.04	11.70	51.79	18.50	-22.76	16.90	18.40	3.22	4
800 mg/kg b.w. test substance 1st experiment	invalid dose group due to high mortality of the treated animals
positive control (2-AAF) 1st experiment	74.68	22.82	22.50	7.90	52.18	20.94	52.18	20.94	100
positive control (2-AAF) 2nd experiment	33.38	11.26	21.11	9.38	12.27	10.19	16.05	8.43	76

SD=Standard deviation. The standard deviation shown for each animal is the deviation between the 100 analysed cells. The deviation shown for the mean of each group is the mean of the standard deviation obtained for each animal for a group consisting of four animals (* three animals) (test item groups) or two animals (control groups).

Group means of nucleus, cytoplasmic area and net grains of females

	Nuclear Grain Count		Cytoplasmic Grain Count		Net Grain Counts		Nuclear Grain Counts of Cells in Repair		Cells in Repair
Test Group	Mean	SD	Mean	SD	Mean	SD	Mean	SD	%
	4 h Preparation Interval
Vehicle control (CMC 1 %)	10.24	4.54	23.17	6.64	-12.93	7.27	2.56	1.25	2
250 mg/kg b.w. test substance	13.69	5.84	22.95	7.83	-9.26	8.06	8.22	1.81	4
500 mg/kg b.w. test substance	16.36	6.67	28.68	9.41	-12.32	9.60	5.52	1.77	4
positive control (DMH)	65.65	16.83	19.12	6.40	46.54	16.61	46.72	16.44	100
	16 h Preparation Interval
Vehicle control (CMC 1 %)	17.83	9.07	36.21	12.59	-18.38	10.96	2.75	0.50	2
250 mg/kg b.w. test substance	18.11	8.04	31.56	10.63	-13.45	10.91	8.44	1.56	4
500 mg/kg b.w. test substance*	16.80	7.14	30.36	11.17	-13.56	10.09	4.92	1.06	3
positive control (2-AAF)	31.83	12.31	27.88	10.27	3.95	10.78	13.48	7.37	44

SD=Standard deviation. The standard deviation shown for each animal is the deviation between the 100 analysed cells. the deviation shown for the mean of each group is the mean of the standard deviations obtained for each animal for a group consisting of four animals (* three animals) (test item groups) or two animals (control groups).

Viability and number of hepatocytes for the males, 4 h timepoint

Treatment	Period	Animal no.	Viability*[%]	Number of isolated cells [x10^6]
1 % CMC (1^stmain experiment)	4 h	1	62	158
		2	78	351
1 % CMC (2^ndmain experiment)	4 h	49	69	267
		50	78	246
200 mg/kg b.w. Test substance	4 h	51	89	318
		52	77	244
		53	76	211
		54	78	160
400 mg/kg b.w. Test substance	4 h	3	81	88.5
		4	68	233
		5	73	210
		6	Perfusion failed
800 mg/kg b.w. Test substance	4 h	7	62	212
		8	65	374
		9	70	186
		10	68	281
80 mg/kg b.w. DMH (1^stmain experiment)	4 h	11	75	255
		12	85	302
80 mg/kg b.w. DMH (2^ndmain experiment)	4 h	55	78	265
		56	75	233

* Viability determined by means of trypan blue dye exclusion assay

Viability and number of hepatocytes for the males, 16 h timepoint

Treatment	Period	Animal no.	Viability*[%]	Number of isolated cells [x10^6]
1 % CMC (1^stmain experiment)	16 h	13	72	306
		14	90	266
1 % CMC (2^ndmain experiment)	16 h	57	83	251
		58	80	228
200 mg/kg b.w. Test substance	16 h	59	82	223
		60	81	134
		61	78	425
		62	73	226
400 mg/kg b.w. Test substance	16 h	15	90	232
		16	82	215
		17	83	255
		18	88	282
800 mg/kg b.w. Test substance	16 h	19	Invalid dose group due to high mortality of the treated animals
		20
		21
		22
100 mg/kg b.w. 2-AAF (1^stmain experiment)	16 h	23	86	275
		24	83	278
100 mg/kg b.w. 2-AAF (2^ndmain experiment)	16 h	63	75	306
		64	65	258

* Viability determined by means of trypan blue dye exclusion assay

Viability and number of hepatocytes for the females, 4 h timepoint

Treatment	Period	Animal no.	Viability*[%]	Number of isolated cells [x 10^6]
1 % CMC	4 h	25	81	75
		26	71	147
250 mg/kg b.w. Test substance	4 h	27	57	85.5
		28	78	318
		29	59	103
		30	79	154
500 mg/kg b.w. Test substance	4 h	31	72	205
		32	82	269
		33	74	196
		34	65	83
80 mg/kg b.w. DMH	4 h	35	90	196
		36	80	270

* Viability determined by means of trypan blue dye exclusion assay

Viability and number of hepatocytes for the females, 16 h timepoint

Treatment	Period	Animal no.	Viability*[%]	Number of isolated cells [´ 10^6]
1 % CMC	16 h	37	85	230
		38	75	174
250 mg/kg b.w. Test substance	16 h	39	85	159
		40	79	142
		41	80	130
		42	80	128
500 mg/kg b.w. Test substance	16 h	43	81	152
		44	Perfusion failed
		45	62	174
		46	80	162
100 mg/kg b.w. 2-AAF	16 h	47	75	203
		48	66	127

* Viability determined by means of trypan blue dye exclusion assay

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the experimental conditions, i.e. oral administration up to 800 mg/kg for males and up to 500 mg/kg for females, the test item did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats. Therefore, the substance is considered to be non-genotoxic in this in vivo UDS test system.

Executive summary:

The substance was assessed under GLP in this valid in vivo UDS assay for its potential to induce unscheduled DNA repair (UDS) in the hepatocytes of rats according to OECD TG 486 with doses of 200, 400 and 800 mg/kg bw for males and 250 and 500 mg/kg bw for females after post treatment intervals of 4 and 16 hours. The highest dose, i.e. the maximum tolerated dose leading to acceptable clinical symptoms, was established in two pre-experiments. The test item was suspended in 1% carboxymethylcellulose, which was also used as the vehicle control. The volume administered orally was 15 mL/kg body weight. After single oral treatment and a post-treatment period of 4 or 16 hours the animals were sacrificed by terminal anaesthesia. The livers were then perfused. Primary hepatocytes wee established and exposed for 4 hours to 3HTdR, which is incorporated if UDS occurs.

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test substance at any of the treatment periods or dose groups. The interindividual variations obtained for the numbers and the viability of the isolated hepatocytes were in the range of the laboratory historical control data. All viabilities were greater than the OECD TG recommended viability of > 50%.

No UDS induction in the hepatocytes of the male and female rats treated with a single oral dose of the test substance as compared to the animals of the concurrent vehicle controls was observed at 200, 400 and 800 mg/kg bw for male rats and at 250 and 500 mg/kg bw for female rats. The nuclear grain counts and the resulting net grain counts were not distinctly enhanced due to the in vivo treatment of the animals with the test item after 4 hours and 16 hours post treatment. Therefore, the mean net grain counts obtained after treatment with the the substance were consistently negative. As no UDS response was observed statistical analysis of the data was not performed. No substantial shift to higher values was obtained in the percentage of cells in repair, which confirms the lack of UDS.

Appropriate reference mutagens (N,N'-dimethylhydrazinedihydrochloride at 80 mg/kg bw and 2-acetylaminofluorene at 100 mg/kg bw) produced distinct increases in the number of nuclear and net grain counts, indicating UDS. Additionally, the percentage of cells in repair was significantly increased, which is consistent with a UDS response. The positive control at the 16 hour preparation interval of the female animals showed a mean net grain count of 3.95 being below the optimal value of 5 net grain counts indicating nominally used in this laboratory to indicate a positive result but within the laboratory historical control data range for similar studies and distinctly different to the strongly negative values observed for the negative control groups. Additionally, as 44% of the cells were in repair (compared to 1.5% of the cells in the vehicle control) the positive control response is considered as valid.