(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted: 21' July 1997, OECD/OCDE Publication Service, Paris

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:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

EC Number:

224-167-6

EC Name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

4221-98-1

Molecular formula:

C10H16

IUPAC Name:

(5R)-2-methyl-5-(propan-2-yl)cyclohexa-1,3-diene

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix induced with Aroclor 1254

Test concentrations with justification for top dose:

5- 5000 µg per plate presence of S9-mix
1.5- 5000 µg per plate absence of S9-mix

Vehicle / solvent:

- Vehicle used: Ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours at 37°C

NUMBER OF REPLICATIONS: The experiment was repeated in full after an interval of at least 3 days.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of bacteriotoxicity.
According Ames et al. (1975) as well as reported by De Serres and Shelby (1979).

Statistics:

XE2-test (Mohn and Ellenberger, 1977)

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:

without S9-mix at 50 µg/plate, with S9-mix at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 mix at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the absence of a metabolizing system (Experiment 1)

	S9 mix	TA98	TA100	TA102	TA1535	TA1537
Control 0 µg/plate	-	13	113	273	12	14
Solvent control 0 µg/plate	-	17	101	293	11	12
Test substance in µg/plate
5	-	15	-	277	14	10
15	-	19	108	296	13	11
50	-	19	93	268	9	9
150	-	11	96	131 (T)	6	8
500	-	16	85	99 (T)	8	6
1500	-	13	86	117 (T)	9	8
5000 (P)	-	12	89	-	-	-
NaN3 (0.7 µg/plate)	-	-	314	-	292	-
2-NF (2.5 µg/plate)	-	395	-	-	-	-
9-AA (50 µg/plate)	-	-	-	-	-	381
Mitomycin C (0.15 µg/plate)	-	-	-	1003	-	-

P: precipitation of test compound

T: bacteriotoxic

Solvent control: Ethanol, 50 µL/plate

2-NF: 2-nitrofluorene

9-AA: 9-aminoacridine

Table 2: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the presence of a metabolizing system (Experiment 1)

	S9 mix	TA98	TA100	TA102	TA1535	TA1537
Control 0 µg/plate	+	22	96	294	7	15
Solvent control 0 µg/plate	+	22	101	290	15	15
Test substance in µg/plate
15	+	19	101	278	17	13
50	+	21	97	188	17	12
150	+	23	101	222	15	15
500	+	17	80	209	17	14
1500	+	22	89	226	17	10
5000 (P)	+	16	93	NA	15	13
2-AA (0.8 µg/plate)	+	501	839	683	123
2-AA (1.7µg/plate)		-	-			314

Solvent control: Ethanol, 50 µL/plate

2-AA: 2-aminoanthracene

Table 3: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the absence of a metabolizing system (Experiment 2)

	S9 mix	TA98	TA100	TA1535	TA1537
Control 0 µg/plate	-	23	84	24	8
Solvent control 0 µg/plate	-	23	76	23	10
Test substance in µg/plate
5	-	21	-	-	-
15	-	14	86	20	10
50	-	23	80	27	8
150	-	19	79	21	6
500	-	12	76	20	10
1500	-	18	79	21	5
5000 (P)	-	-	86	22	6 (T)
NaN3 (0.7 µg/plate)	-	-	283	817	-
2-NF (2.5 µg/plate)	-	258		-	-
9-AA (50 µg/plate)	-			-	844

T: bacteriotoxic

Table 4: Induction of revertants (mean of 3 replicates) in S. typhimurium strains in the presence of a metabolizing system (Experiment 2)

	S9 mix	TA98	TA100	TA102	TA1535	TA1537
Control 0 µg/plate	+	28	90	284	12	13
Solvent control 0 µg/plate	+	27	85	285	18	14
Test substance in µg/plate
5	+	-	-	324	-	-
15	+	25	-	337	-	14
50	+	29	85	325	17	13
150	+	15	82	226	17	13
500	+	26	84	247	18	7
1500	+	27	79	-	16	9
5000 (P)	+	28	83	-	14	14
2-AA (0.8 µg/plate)	+	583	511	-	136	-
2-AA (0.9 µg/plate)				747		-
2-AA (1.7µg/plate)				-		233

Table 5: Induction of revertants (mean of 3 replicates) in S. typhimurium strain TA 102 in the absence of a metabolizing system (Experiment 2)

	S9 mix	TA102
Control 0 µg/plate	-	309
Solvent control 0 µg/plate	-	301
Test substance in µg/plate	-	-
1.5	-	286
5	-	298
15	-	281
50	-	140 (T)
150	-	142 (T)
500	-	135 (T)
Mitomycin C (0.15 µg/plate)	-	959

T: bacteriotoxic

Applicant's summary and conclusion

Conclusions:

The test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.
 

Executive summary:

In the GLP and guideline compliant study the test substance was tested in reverse mutation study with the stains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100, TA 102). The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system.

The test substance was dissolved in ethanol and tested in concentrations of 5 to 5000 µg/plate in the presence and of 1.5 to 5000 µg per plate absence of S9.

The test substance was bacteriotoxic in the absence of S9-mix towards the strains TA 102 at 50 µg/plate and towards the strain TA 1537 at 5000 µg/plate.

In the presence of S9-mix the test substance was bacteriotoxic towards the strains TA 102 at 500 µg/plate. Precipitation of the test compound on the plates was observed at 5000 µg/plate.

 

The positive and negative controls were all valid and in the expected ranges.

In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.

In conclusion, these results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.