4-chlorobenzonitrile

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:

07.01.2014 - 06.05.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-chlorobenzonitrile

EC Number:

210-765-4

EC Name:

4-chlorobenzonitrile

Cas Number:

623-03-0

Molecular formula:

C7H4ClN

IUPAC Name:

4-chlorobenzonitrile

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): 4-Chlorobenzonitrile
- Physical state: solid
- Analytical purity: 99.7 %
- Impurities (identity and concentrations): 0.05 % 2-Chlorobenzonitrile; 0.09 % 3-Chlorobenzonitrile; 0.06 % Chlorobenzaldehyde; < 0.05 % 4-Chlorotoluene, Benzonitrile; 0.1 % another organic impurity max.
- Purity test date: 04.09.2013
- Lot/batch No.: 324601
- Expiration date of the lot/batch: 31.12.2015
- Storage condition of test material: Room Temperature (20 +/- 5 °C)

Method

Target gene:

Gene involved in histidine synthesis

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Details on mammalian cell type (if applicable):

n. a.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

Experiment I: 15, 50, 150, 500 and 1500 µg/plate
Experiment II: 94, 188, 375, 750 and 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The test item was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine; 2-Aminoanthracene

Details on test system and experimental conditions:

see "any other information om materials and methods incl. tables"

Evaluation criteria:

The colonies were counted visually and, the numbers were recorded. A spreadsheet soft-ware (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The increase factor f(I) of revertant induction (mean revertants divided by mean spontane-ous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor >= 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

no data

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 97

Remarks:

TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

1 Results

1. 1 First Experiment

1.1.1 Confirmation of the Criteria and Validity

The treatments for the sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.

1.1.2    Solubility and Toxicity

The test item was dissolved in DMSO. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.

1.1.3    Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

In the treatment with metabolic activation in the highest concentration (1500 µg/plate) the mean value of the revertants of the strain TA97a is higher than the mean value of the spontaneous revertants of the solvent control DMSO. But it lies below the threshold of the increase factor for mutagenicity. Furthermore, in the second experiment, the respective value clearly shows no evidence of mutageniticty. Bacteria strains are living biological systems, therefore variations in behaviour are not unusual.

The mean revertant values of the four replicates are presented in table 1.

Table 1: Mean Revertants of the First Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	166	101	25	30	93	93	329	169	15	13
	sd	8.5	21.2	9.3	4.9	27.1	11.4	18.6	30.0	1.7	1.4
DMSO	Mean	150	97	23	24	167	134	173	331	12	15
	sd	7.6	10.8	2.6	9.6	9.1	52.8	25.6	78.6	1.3	1.4
Positive Controls*	Mean	511	225	194	248	290	307	417	1148	180	195
	sd	71	40	29	47	53	30	52	45	29	48
	f(I)	3.41	2.32	8.43	10.33	3.12	2.29	2.41	3.47	12.00	13.00
1500 µg/plate	Mean	138	189	15	24	162	149	280	198	14	12
	sd	23	23	4	5	20	37	61	5	3	2
	f(I)	0.92	1.95	0.65	1.00	0.97	1.11	1.62	0.60	1.17	0.80
500 µg/plate	Mean	140	140	22	25	144	102	266	270	15	16
	sd	29	19	4	10	32	22	32	10	3	3
	f(I)	0.93	1.44	0.96	1.04	0.86	0.76	1.54	0.82	1.25	1.07
150 µg/plate	Mean	110	133	23	24	158	97	245	216	15	17
	sd	13	39	7	8	13	11	40	18	3	2
	f(I)	0.73	1.37	1.00	1.00	0.95	0.72	1.42	0.65	1.25	1.13
50 µg/plate	Mean	125	151	23	23	170	160	281	253	16	17
	sd	27	33	4	5	7	29	32	39	5	5
	f(I)	0.83	1.56	1.00	0.96	1.02	1.19	1.62	0.76	1.33	1.13
15 µg/plate	Mean	121	137	17	22	130	117	249	230	14	17
	sd	35	18	2	6	37	27	38	29	4	1
	f(I)	0.81	1.41	0.74	0.92	0.78	0.87	1.44	0.69	1.17	1.13

sd = standard deviation; f(I) = increase factor; * = Different positive controls were used

1.2     Second Experiment

1.2.1    Confirmation of the Criteria and Validity

The treatments for the sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls showed mutagenic effects with and without metabolic activation.

1.2.2    Solubility and Toxicity

The test item was dissolved in DMSO. No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not significantly reduced.

1.2.3    Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found. Therefore, the test item is stated as not mutagenic under the test conditions.

The mean revertant values of the four replicates are presented in table 2.

Table 2: Mean Revertants of the Second Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	107	117	25	25	85	125	306	291	15	15
	sd	4.3	4.7	3.8	2.5	11.7	12.8	39.0	16.5	4.9	2.4
DMSO	Mean	119	125	27	24	99	105	317	284	14	17
	sd	6.7	11.2	4.7	3.8	20.6	5.3	45.8	16.7	1.6	1.7
Positive Controls*	Mean	440	418	412	212	587	413	1046	820	162	121
	sd	13	27	8	28	99	13	182	45	35	20
	f(I)	3.70	3.34	15.26	8.83	6.91	3.93	3.30	2.89	10.80	7.12
1500 µg/plate	Mean	122	122	14	24	156	90	254	266	16	13
	sd	2	16	9	3	13	13	24	10	4	2
	f(I)	1.03	0.98	0.52	1.00	1.58	0.86	0.80	0.94	1.14	0.76
750 µg/plate	Mean	120	134	21	32	171	98	266	312	16	15
	sd	3	11	4	10	16	12	11	80	4	3
	f(I)	1.01	1.07	0.78	1.33	1.73	0.93	0.84	1.10	1.14	0.88
375 µg/plate	Mean	121	123	23	27	123	137	304	278	16	17
	sd	3	40	5	2	29	16	39	14	3	3
	f(I)	1.02	0.98	0.85	1.13	1.24	1.30	0.96	0.98	1.14	1.00
188 µg/plate	Mean	141	109	22	24	98	135	298	297	15	17
	sd	29	37	4	5	11	6	26	25	4	4
	f(I)	1.18	0.87	0.81	1.00	0.99	1.29	0.94	1.05	1.07	1.00
94 µg/plate	Mean	114	134	21	26	105	140	303	294	13	22
	sd	8	35	5	5	14	10	14	26	3	2
	f(I)	0.96	1.07	0.78	1.08	1.06	1.33	0.96	1.04	0.93	1.29

sd = standard deviation; f(I) = increase factor; * = Different positive controls were used

Applicant's summary and conclusion

Executive summary:

In a study according to OECD 471 and EU Method B.13/14 two valid experiments were performed.

First Experiment:

Five concentrations of the test item, dissolved in DMSO were used. Five genetically manipulated strains of Salmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method.

None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item didn’t show any mutagenic effects in the first experiment. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second Experiment:

To verify the results of the first experiment, a second experiment was performed, using five concentrations of the test item and a modification in study performance (pre-incubation method).

The test item didn’t show mutagenic effects in the second experiment, either. No signs of toxicity towards the bacteria could be observed. The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

 

Under the conditions of the test, the test item didn’t show mutagenic effects towards Salmonella typhimurium strainsTA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined.

 

The test item 4-Chlorobenzonitrile is considered as “not mutagenic under the conditions of the test”.