2-nitro-4-(trifluoromethyl)benzonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 11 Mar 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 26 Jun 2020

Deviations:

yes

Remarks:

Pre-test data for solvent and cytotoxicity testing not included in study report.

GLP compliance:

yes

Remarks:

The test complied with the Principles of Good Laboratory Practices (GLP) of the Certification and Accreditation Administration of the People’s Republic of China (2013 revised edition).

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-nitro-4-(trifluoromethyl)benzonitrile

EC Number:

212-298-1

EC Name:

2-nitro-4-(trifluoromethyl)benzonitrile

Cas Number:

778-94-9

Molecular formula:

C8H3F3N2O2

IUPAC Name:

2-nitro-4-(trifluoromethyl)benzonitrile

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97a

Additional strain / cell type characteristics:

other: Requirements met in histidine dependence test, lipopolysaccharide barrier defect test, ultraviolet ray injury repair defect test, ampicillin/tetracycline resistance test, diagnostic test, spontaneous reverse mutation test

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: Requirements met in histidine dependence test, lipopolysaccharide barrier defect test, ultraviolet ray injury repair defect test, ampicillin/tetracycline resistance test, diagnostic test, spontaneous reverse mutation test

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: Requirements met in histidine dependence test, lipopolysaccharide barrier defect test, ultraviolet ray injury repair defect test, ampicillin/tetracycline resistance test, diagnostic test, spontaneous reverse mutation test

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: Requirements met in histidine dependence test, lipopolysaccharide barrier defect test, ultraviolet ray injury repair defect test, ampicillin/tetracycline resistance test, diagnostic test, spontaneous reverse mutation test

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: Requirements met in histidine dependence test, lipopolysaccharide barrier defect test, ultraviolet ray injury repair defect test, ampicillin/tetracycline resistance test, diagnostic test, spontaneous reverse mutation test

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix)
- concentration or volume of S9 mix and S9 in the final culture medium: 10%

Test concentrations with justification for top dose:

0.5, 1.581, 5, 15.81 and 50 µg/plate for all strains with and without metabolic activation.
The doses were selected based on the results of a pre-test with doses between 10 and 5000 µg/plate, in which bacterial toxicity was observed in all apart from the lowest concentration tested. Furthermore, slight precipitation was noted at 5000 µg/plate (-S9 conditions) only prior to the start of culturing. No precipitation occurred at any other dose group.

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: DMSO was used as solvent in the pre-test, therefore, DMSO was also selected as solvent for the main experiment.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other:

Remarks:

all positive controls dissolved in DMSO
1) TA97a, TA98, TA102: 50 µg/plate (-S9)
2) TA97a, TA98, TA100: 20 µg/plate (+S9)
3) TA102: 50 µg/plate (+S9)
4) TA1535: 2 µg/plate (+S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
3 plates for all dose groups and control groups in 2 independent experiments.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h (first experiment), 64 h (second experiment)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate

Rationale for test conditions:

According to "Chemical Registration Centre of the Ministry of Environmental Protection: Chemical Test Methods – Health Effects Volume – 471 Bacterial Reverse Mutation Test [M]. Second edition. Beijing: China Environment Press, 2013: 410-416".

Evaluation criteria:

In a metabolically activated or not metabolically activated system, when the revertant bacterial strain count in the test sample group compared to the solvent control group exceeded a certain numerical range (that is, in the test bacteria TA1535 test sample group, the revertant bacterial strain count was equal to or exceeded the mean of the solvent control group by a factor of 3; in the other test bacteria test sample groups, the revertant bacterial strain count was equal to or exceeded the solvent control group by a factor of 2), and a dose-response relationship existed among all doses, or at a certain test point (bacterial strain or dose) the increase in revertant bacterial colony count was reproducible, then the test results could be judged positive. Otherwise, the test results were negative.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

S9(-): ≥ 15.18 µg/plate S9(+): 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

S9(-): 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

S9(-): 50 µg/plate S9(+): 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

S9(-): ≥ 15.18 µg/plate, S9(+): 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

S9(-): 15.18 µg/plate S9 (+): 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: no precipitation observed

RANGE-FINDING/SCREENING STUDIES
In the pre-test, DMSO was used as the solvent. A total of seven doses were established: 5000, 1000, 300, 100, 60, 30, and 10 μg/plate. Under not metabolically activated conditions, the plate incorporation test was carried out on the test bacterial strains TA97a, TA98, TA100, TA102, and TA1535, two plates per group, and a simultaneous solvent control was established. The results of the pre-test revealed that under not metabolically activated conditions, at 5000 μg/plate dose prior to the start of culturing, there was slight precipitation, but after the end of culturing, there was no precipitation. There were no signs of precipitation for the other dose groups. For the 5000, 1000, and 300 μg/plate doses, bacterial colonies died and there was high bacterial toxicity. For the 100 and 60 μg/plate doses, bacterial colonies died or were notably reduced and there was high toxicity or moderate toxicity. For bacterial strains TA98 and TA1535 at the 30 μg/plate dose, bacterial colonies were notably reduced and there was bacterial toxicity. For the 10 μg/plate dose, there was no obvious bacterial toxicity.

STUDY RESULTS
No indication of genotoxicity was observed in any bacterial strain. For details, please refer to Tables 1 - 6 under section "Any other information on results incl. tables".


HISTORICAL CONTROL DATA: not reported

Remarks on result:

other: not applicable

Any other information on results incl. tables

Table 1: Results of the first experiment (individual values)

μg/plate	No.	S9-					S9+
		TA97a	TA98	TA100	TA102	TA1535	TA97a	TA98	TA100	TA102	TA1535
Solvent control	1	183	13	108	310	14	197	24	119	274	19
	2	165	16	106	296	8	199	22	121	259	20
	3	158	16	124	316	8	186	27	143	265	24
50	1	51	2	0	256	0	180	8	32	211	0
	2	49	5	0	241	0	170	17	26	207	0
	3	58	3	0	220	0	179	16	33	223	0
15.81	1	201	18	73	260	3	211	18	168	282	15
	2	194	16	62	274	4	195	14	153	261	13
	3	211	20	85	225	6	178	22	134	258	16
5	1	147	14	102	320	9	155	23	123	257	16
	2	131	18	103	357	6	100	18	125	275	12
	3	193	14	90	297	8	179	17	143	257	16
1.581	1	161	20	125	286	12	149	18	124	201	10
	2	154	27	101	344	6	154	19	146	222	22
	3	145	12	103	314	13	139	22	138	243	17
0.5	1	176	26	98	293	15	233	25	157	266	19
	2	185	19	151	298	10	232	15	156	257	16
	3	150	18	80	296	12	213	28	165	281	25
Positive control	1	3200	1132	882	1346	229	1128	846	1026	986	326
	2	2974	1008	768	1228	227	1360	886	1148	942	348
	3	2624	1184	776	1264	253	1208	986	1266	884	326

 

 

Table 2: Results of the first experiment without metabolic activation (mean values)

μg/plate	Statistical results	S9-
		TA97a	TA98	TA100	TA102	TA1535
Solvent control	Mean ± SD Ratio	168.67 ± 12.90 1.00	15.00 ± 1.73 1.00	112.67 ± 9.87 1.00	307.33 ± 10.26 1.00	10.00 ± 3.46 1.00
50	Mean ± SD Ratio	52.67 ± 4.73 0.31	3.33 ± 1.53 0.22	0.00 ± 0.00 0.00	239.00 ± 18.08 0.78	0.00 ± 0.00 0.00
15.81	Mean ± SD Ratio	202.00 ± 8.54 1.20	18.00 ± 2.00 1.20	73.33 ± 11.50 0.65	253.00 ± 25.24 0.82	4.33 ± 1.53 0.43
5	Mean ± SD Ratio	157.00 ± 32.19 0.93	15.33 ± 2.31 1.02	98.33 ± 7.23 0.87	324.67 ± 30.27 1.06	7.67 ± 1.53 0.77
1.581	Mean ± SD Ratio	153.33 ± 8.02 0.91	19.67 ± 7.51 1.31	109.67 ± 13.32 0.97	314.67 ± 29.01 1.02	10.33 ± 3.79 1.03
0.5	Mean ± SD Ratio	170.33 ± 18.18 1.01	21.00 ± 4.36 1.40	109.67 ± 36.91 0.97	295.67 ± 2.52 0.96	12.33 ± 2.52 1.23
Positive control	Mean ± SD Ratio	2932.67 ± 290.22 17.39	1108.00 ± 90.42 73.87	808.67 ± 63.63 7.18	1279.33 ± 60.48 4.16	236.33 ± 14.47 23.63

 

 

 

Table 3: Results of the first experiment with metabolic activation (mean values)

μg/plate	Statistical results	S9+
		TA97a	TA98	TA100	TA102	TA1535
Negative control	Mean ± SD Ratio	194.00 ± 7.00 1.00	24.33 ± 2.52 1.00	127.67 ± 13.32 1.00	266.00 ± 7.55 1.00	21.00 ± 2.65 1.00
50	Mean ± SD Ratio	176.33 ± 5.51 0.91	13.67 ± 4.93 0.56	30.33 ± 3.79 0.24	213.67 ± 8.33 0.80	0.00 ± 0.00 0.00
15.81	Mean ± SD Ratio	194.67 ± 16.50 1.00	18.00 ± 4.00 0.74	151.67 ± 17.04 1.19	267.00 ± 13.08 1.00	14.67 ± 1.53 0.70
5	Mean ± SD Ratio	144.67 ± 40.50 0.75	19.33 ± 3.21 0.79	130.33 ± 11.02 1.02	263.00 ± 10.39 0.99	14.67 ± 2.31 0.70
1.581	Mean ± SD Ratio	147.33 ± 7.64 0.76	19.67 ± 2.08 0.81	136.00 ± 11.14 1.07	222.00 ± 21.00 0.83	16.33 ± 6.03 0.78
0.5	Mean ± SD Ratio	226.00 ± 11.27 1.16	22.67 ± 6.81 0.93	159.33 ± 4.93 1.25	268.00 ± 12.12 1.01	20.00 ± 4.58 0.95
Positive control	Mean ± SD Ratio	1232.00 ± 117.85 6.35	906.00 ± 72.11 37.23	1146.67 ± 120.01 8.98	937.33 ± 51.16 3.52	333.33 ± 12.70 15.87

 

 

Table 4: Results of the second experiment (individual values)

μg/plate	No.	S9-					S9+
		TA97a	TA98	TA100	TA102	TA1535	TA97a	TA98	TA100	TA102	TA1535
Solvent control	1	212	23	139	248	21	207	28	155	345	36
	2	196	29	104	278	34	205	28	157	314	29
	3	191	23	127	295	30	192	24	143	327	24
50	1	60	4	0	198	0	277	15	77	194	3
	2	57	3	0	194	0	245	20	97	203	4
	3	40	3	0	224	0	288	17	100	212	6
15.81	1	247	21	94	216	5	302	21	199	260	30
	2	204	28	87	265	9	297	17	205	257	27
	3	201	27	105	222	24	245	24	182	249	29
5	1	189	19	147	263	24	291	27	137	300	28
	2	184	23	144	222	25	278	28	137	266	27
	3	196	31	135	236	16	263	31	142	278	32
1.581	1	199	19	96	237	29	273	23	118	250	28
	2	169	23	143	252	22	248	22	151	231	45
	3	181	20	114	244	16	232	25	145	268	32
0.5	1	171	24	136	281	20	220	34	214	266	43
	2	164	30	103	257	27	218	30	201	282	30
	3	209	19	98	260	25	209	29	222	270	29
Positive control	1	3488	872	606	927	364	1102	678	789	1128	223
	2	3168	1064	768	836	403	987	658	897	1287	307
	3	3296	928	559	1024	375	1036	712	778	996	287

 

 

Table 5: Results of the second experiment without metabolic activation (mean values)

μg/plate	Statistical results	S9-
		TA97a	TA98	TA100	TA102	TA1535
Negative control	Mean ± SD Ratio	199.67 ± 10.97 1.00	25.00 ± 3.46 1.00	123.33 ± 17.79 1.00	273.67 ± 23.80 1.00	28.33 ± 6.66 1.00
50	Mean ± SD Ratio	52.33 ± 10.79 0.26	3.33 ± 0.58 0.13	0.00 ± 0.00 0.00	205.33 ± 16.29 0.75	0.00 ± 0.00 0.00
15.81	Mean ± SD Ratio	217.33 ± 25.74 1.09	25.33 ± 3.79 1.01	95.33 ± 9.07 0.77	234.33 ± 26.73 0.86	12.67 ± 10.02 0.45
5	Mean ± SD Ratio	189.67 ± 6.03 0.95	24.33 ± 6.11 0.97	142.00 ± 6.24 1.15	240.33 ± 20.84 0.88	21.67 ± 4.93 0.76
1.581	Mean ± SD Ratio	183.00 ± 15.10 0.92	20.67 ± 2.08 0.83	117.67 ± 23.71 0.95	244.33 ± 7.51 0.89	22.33 ± 6.51 0.79
0.5	Mean ± SD Ratio	181.33 ± 24.21 0.91	24.33 ± 5.51 0.97	112.33 ± 20.65 0.91	266.00± 13.08 0.97	24.00 ± 3.61 0.85
Positive control	Mean ± SD Ratio	3317.33 ± 161.06 16.61	954.67 ± 98.74 38.19	644.33 ± 109.65 5.22	929.00 ± 94.02 3.39	380.67 ± 20.11 13.44

 

 

Table 6: Results of the second experiment with metabolic activation (mean values)

μg/plate	Statistical results	S9+
		TA97a	TA98	TA100	TA102	TA1535
Negative control	Mean ± SD Ratio	201.33 ± 8.14 1.00	26.67 ± 2.31 1.00	151.67 ± 7.57 1.00	328.67 ± 15.57 1.00	29.67 ± 6.03 1.00
50	Mean ± SD Ratio	270.00 ± 22.34 1.34	17.33± 2.52 0.65	91.33 ± 12.50 0.60	203.00 ± 9.00 0.62	4.33 ± 1.53 0.15
15.81	Mean ± SD Ratio	281.33 ± 31.56 1.40	20.67 ± 3.51 0.78	195.33 ± 11.93 1.29	255.33 ± 5.69 0.78	28.67 ± 1.53 0.97
5	Mean ± SD Ratio	277.33 ± 14.01 1.38	28.67 ± 2.08 1.08	138.67 ± 2.89 0.91	281.33 ± 17.24 0.86	29.00 ± 2.65 0.98
1.581	Mean ± SD Ratio	251.00 ± 20.66 1.25	23.33 ± 1.53 0.88	138.00 ± 17.58 0.91	249.67 ± 18.50 0.76	35.00 ± 8.89 1.18
0.5	Mean ± SD Ratio	215.67 ± 5.86 1.07	31.00 ± 2.65 1.16	212.33 ± 10.60 1.40	272.67 ± 8.33 0.83	34.00 ± 7.81 1.15
Positive control	Mean ± SD Ratio	1041.67 ± 57.71 5.17	682.67 ± 27.30 25.60	821.33 ± 65.76 5.42	1137.00 ± 145.71 3.46	272.33 ± 43.88 9.18

 

Applicant's summary and conclusion

Conclusions:

The test results indicated that, under the conditions of this test, 2-nitro-4-(trifluoromethyl)benzonitrile did not have mutagenicity on the test bacteria.