2,2,3-trimethylcyclopent-3-enylacetonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-06-19 to 2015-06-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD Guideline and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2,3-trimethylcyclopent-3-enylacetonitrile

EC Number:

239-405-4

EC Name:

2,2,3-trimethylcyclopent-3-enylacetonitrile

Cas Number:

15373-31-6

Molecular formula:

C10H15N

IUPAC Name:

2-(2,2,3-trimethylcyclopent-3-en-1-yl)acetonitrile

Method

Target gene:

Salmonella typhimurium: histidine locus
Escherichia coli: tryptophan locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(NaN3), 10 μg/plate, without metabolic activation TA 1535, TA 100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

(4-NOPD), without metabolic activation, 10 μg/plate in strain TA 98, 50 μg/plate in strain TA 1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

(MMS), 2.0 μL/plate, without metabolic activation, WP2 uvrA

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(2-AA), with metabolic activation, TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

A test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed.
A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase was not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data was not mandatory. Therefore no analysis was performed.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in the presence of S9 mix

ADDITIONAL INFORMATION ON CYTOTOXICITY:
experiment I: without S9: TA1537 (5000 μg/plate), TA100 (2500-5000 μg/plate), WP2uvrA (5000 μg/plate); with S9 mix: TA1535 and TA100 (1000-5000 μg/plate), TA1537 and TA98 and WP2uvrA (2500-5000 μg/plate)
experiment II: seen for all strains with and without S9 mix: 1000-5000 μg/plate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Plate incorporation test (experiment 1):

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results without S9
DMSO	9 ± 1	9 ± 4	19 ± 6	147 ± 8	46 ± 8
Untreated	8 ± 2	9 ± 4	25 ± 2	152 ± 17	53 ± 1
3	10 ± 3	9 ± 2	21 ± 7	166 ± 21	49 ± 9
10	9 ± 4	8 ± 2	24 ± 7	154 ± 7	42 ± 6
33	14 ± 2	7 ± 2	25 ± 4	151 ± 17	47 ± 4
100	8 ± 2	7 ± 1	26 ± 5	150 ± 19	47 ± 3
333	13 ± 4	5 ± 0	23 ± 5	90 ± 19	43 ± 6
1000	15 ± 6	7 ± 1	17 ± 5	67 ± 3	33 ± 5
2500	11 ± 3	7 ± 1	17 ± 6	58 ± 1	23 ± 2
5000	11 ± 5	2 ± 2	14 ± 2	63 ± 10	16 ± 3
NaN3 (10)	1205 ± 87			2027 ± 45
4-NOPD (10)			327 ± 17
4-NOPD (50)		72 ± 12
MMS (2.0 µL)					985 ± 46
	Results with S9
DMSO	13 ± 1	8 ± 0	27 ± 5	135 ± 1	58 ± 8
Untreated	13 ± 4	13 ± 3	33 ± 2	149 ± 4	63 ± 12
3	11 ± 1	9 ± 4	30 ± 5	144 ± 30	56 ± 5
10	11 ± 3	11 ± 3	27 ± 7	134 ± 16	60 ± 9
33	15 ± 2	12 ± 2	35 ± 3	150 ± 20	56 ± 6
100	12 ± 0	13 ± 1	27 ± 6	150 ± 28	66 ± 14
333	8 ± 3	15 ± 2	35 ± 2	110 ± 5	61 ± 17
1000	6 ± 2	4 ± 2	23 ± 1	57 ± 13	29 ± 9
2500	6 ± 2	1 ± 1	4 ± 1	19 ± 3	9 ± 3
5000	1 ± 1	0 ± 0	0 ± 0	0 ± 1	1 ± 1
2-AA (2.5)	446 ± 22	120 ± 9	3437 ± 519	3362 ± 163
2-AA (10.0)					423 ± 41

Pre-incubation test (experiment 2:

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and E. coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results without S9
DMSO	15 ± 1	10 ± 4	20 ± 4	118 ± 7	43 ± 6
Untreated	11 ± 4	10 ± 2	21 ± 4	148 ± 20	37 ± 1
3	8 ± 3	8 ± 2	21 ± 2	131 ± 10	37 ± 4
10	7 ± 1	8 ± 2	27 ± 1	129 ± 9	35 ± 8
33	11 ± 2	8 ± 4	18 ± 2	124 ± 12	41 ± 9
100	9 ± 2	9 ± 2	24 ± 3	106 ± 29	48 ± 8
333	9 ± 4	7 ± 2	21 ± 5	56 ± 5	38 ± 6
1000	4 ± 2	1 ± 1	0 ± 1	26 ± 23	13 ± 7
2500	2 ± 2	0 ± 0	0 ± 0	11 ± 11	11 ± 8
5000	0 ± 0	0 ± 0	0 ± 0	5 ± 8	0 ± 0
NaN3 (10)	1201 ± 21			2191 ± 176
4-NOPD (10)			338 ± 16
4-NOPD (50)		92 ± 4
MMS (2.0 µL)					608 ± 17
	Results with S9
DMSO	11 ± 2	13 ± 1	37 ± 7	105 ± 14	48 ± 3
Untreated	9 ± 3	9 ± 1	39 ± 9	146 ± 8	42 ± 9
3	8 ± 1	11 ± 2	32 ± 3	101 ± 11	43 ± 8
10	7 ± 2	12 ± 5	33 ± 1	114 ± 5	51 ± 5
33	10 ± 4	15 ± 5	32 ± 3	108 ± 10	57 ± 6
100	13 ± 1	17 ± 3	36 ± 2	108 ± 5	54 ± 14
333	9 ± 4	13 ± 3	33 ± 4	63 ± 8	48 ± 1
1000	0 ± 1	1 ± 1	1 ± 1	2 ± 3	21 ± 5
2500	0 ± 1	0 ± 0	0 ± 0	0 ± 0	0 ± 1
5000	0 ± 0	0 ± 0	0 ± 0	0 ± 0	0 ± 0
2-AA (2.5)	420 ± 26	134 ± 18	4232 ± 148	3123 ± 114
2-AA (10.0)					410 ± 17

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

An in vitro reverse mutation assay study (Ames) was conducted in Salmonella typhimurium and Escherichia coli strains. It was concluded that the test substance did not induce gene mutations and was considered to be non-mutagenic.

Executive summary:

An in vitro reverse mutation assay study (Ames) was conducted according to OECD Guideline 471 in Salmonella typhimurium and Escherichia coli strains. Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay. All tests were conducted in triplicate and concentrations between 3 μg/plate and 5000 μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Cytotoxicity was observed in the plate incorporation test without S9 in three strains (TA1537 (5000 μg/plate), TA100 (2500-5000 μg/plate), WP2uvrA (5000 μg/plate)) and with S9 mix in all strains (TA1535 and TA100 (1000-5000 μg/plate), TA1537 and TA98 and WP2uvrA (2500-5000 μg/plate)). In the pre-incubation test cytotoxicity was observed in all test strains independent of metabolic activation in the range from 1000 to 5000 μg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was detected following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore it was concluded that the test substance did not induce gene mutations and it was considered to be non-mutagenic.