Reaction mass of [(1R,2S)-2-cyclopentylcyclopentyl] (E)-but-2-enoate and [(1S,2R)-2-cyclopentylcyclopentyl] (E)-but-2-enoate and [(1S,2S)-2-cyclopentylcyclopentyl] (E)-but-2-enoate and [(1R,2R)-2-cyclopentylcyclopentyl] (E)-but-2-enoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro: Ames test (OECD 471): negative with S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and E. coli WP2 uvrA with and without metabolic activation

Link to relevant study records

Reference

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:

10 July - 10 Aug 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

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)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone (80/100 mg/kg bw/day for 3 days)

Test concentrations with justification for top dose:

Range-finding study:
TA100 and WP2 uvrA: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment 1: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment 2:
TA100, TA98, TA1537 and WP2 uvrA: 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation
TA1535: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation
TA100 and WP2 uvrA: 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation
TA1535, TA98 and TA1537: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was incompatible with water but was fully soluble in DMSO at 50 mg/mL.

Untreated negative controls:

yes

Remarks:

without S9 mix

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene (2-AA)

Remarks:

+S9: 2-AA (1 µg/plate, TA100; 2 µg/plate, TA1535, TA1537; 10 µg/plate, WP2 uvrA); B[a]P (5 µg/plate, TA98); -S9: ENNG (2 µg/plate, WP2 uvrA; 3 µg/plate, TA100; 5 µg/plate, TA1535); 9-AA (80 µg/plate, TA1537); 4-NQO (0.2 µg/plate, TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

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

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods can also be used as an aid to evalutation, however, statistical significance will not be the only determining factor for a positive response.
A test substance will be considered non-mutagenic in the test system if the above criteria are not met.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(exp. 2: -S9: starting at 500 µg/plate in TA 1535; +S9: starting at 1500 µg/plate in TA 1535 and at 5000 µg/plate in TA 98 and TA 1537)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to the precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: An oily precipitate was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for the main study, a range-finding study was carried out using TA 100 and WP2 uvrA. Test substance concentrations from 0.15 to 5000 µg/plate were tested with and without metabolic activation. The test substance was non-toxic to the tested strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In experiment 1, the test material caused no visible reduction in the growth of the bacterial background lawns at any dose level either with or without S9 mix. In experiment 2, the test substance caused a visible reduction in the growth of the bacterial background lawns and/or substantial decrease in the revertant colony frequency of several of the strains both with and without metabolic activation, initially at 500 µg/plate. The results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	WP2 uvrA	TA98	TA1537
–	0	95	18	22	17	12
–	0 (DMSO)	98 ± 1.2	16 ± 1.7	24 ± 2.3	19 ± 3.5	14 ± 0.6
–	50	93 ± 10.3	14 ± 1.5	23 ± 4.0	18 ± 2.5	14 ± 0.6
–	150	85 ± 5.6	16 ± 2.5	23 ± 3.5	19 ± 3.6	14 ± 2.1
–	500	87 ± 2.3	15 ± 4.0	19 ± 1.5	15 ± 2.3	13 ± 3.1
–	1500	84 ± 5.6	16 ± 3.8	23 ± 6.7	17 ± 4.0	15 ± 2.9
–	5000	90 ± 1.5^P	18 ± 1.5^P	23 ± 4.5^P	16 ± 4.0^P	14 ± 1.2^P
Positive controls, –S9	Name	ENNG	ENNG	ENNG	4-NQO	9-AA
	Concentration [μg/plate]	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	492 ± 29.2	115 ± 30.7	145 ± 0.0	119 ± 4.0	400 ± 58.8
+	0 (DMSO)	90 ± 7.8	12 ± 0.6	21 ± 2.5	24 ± 4.4	13 ± 2.0
+	50	97 ± 6.2	11 ± 3.6	20 ± 1.5	21 ± 3.0	13 ± 1.2
	150	94 ± 4.9	12 ± 1.0	24 ± 4.0	21 ± 1.7	13 ± 2.1
+	500	73 ± 3.2	12 ± 2.3	21 ± 2.9	16 ± 1.5	14 ± 4.0
+	1500	68 ± 6.2	9 ± 3.5	22 ± 3.1	22 ± 4.0	8 ± 1.0
+	5000	68 ± 3.2^P	8 ± 3.0^P	20 ± 0.6^P	12 ± 1.2^P	6 ± 2.1^P
Positive controls, +S9	Name	2-AA	2-AA	2-AA	B[a]P	2-AA
	Concentration [μg/plate]	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	2506 ± 248.1	179 ± 29.1	184 ± 36.9	197 ± 11.7	247 ± 30.5

DMSO: dimethylsulphoxide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

4-NQO: 4-nitroquinoline-N-oxide

B[a]P: benzo(a)pyrene

P: precipitate

Table 2. Test results of experiment 2 (preincubation).

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA100	TA1535	WP2 uvrA	TA98	TA1537
–	0	118	15	29	20	13
–	0 (DMSO)	102 ± 13.1	21 ± 2.0	26 ± 5.9	22 ± 1.2	15 ± 1.5
–	5	n.d.	22 ± 1.5	n.d.	n.d.	n.d.
–	15	n.d.	23 ± 1.5	n.d.	n.d.	n.d.
–	50	89 ± 9.2	25 ± 2.1	24 ± 8.5	23 ± 5.2	12 ± 2.9
–	150	88 ± 10.3	24 ± 2.6	29 ± 3.6	26 ± 0.6	11 ± 3.5
–	500	93 ± 12.3	21 ± 2.1^#	22 ± 1.7	21 ± 1.0	10 ± 5.0
–	1500	84 ± 8.1	19 ± 4.6^#	27 ± 3.6	26 ± 1.2	12 ± 1.5
–	5000	81 ± 3.8^P,#	26 ± 5.8^P,#	24 ± 2.3^P	20 ± 2.5^P	11 ± 2.6^P
Positive controls, –S9	Name	ENNG	ENNG	ENNG	4-NQO	9-AA
	Concentration [μg/plate]	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	309 ± 12.8	432 ± 50.8	462 ± 40.3	228 ± 15.9	1426 ± 535.2
+	0 (DMSO)	107 ± 14.0	13 ± 2.6	24 ± 4.9	19 ± 1.0	12 ± 1.5
+	5	n.d.	15 ± 1.5	n.d.	21 ± 3.0	9 ± 1.7
+	15	n.d.	11 ± 0.6	n.d.	19 ± 1.0	13 ± 3.5
+	50	99 ± 15.9	12 ± 2.3	29 ± 5.8	19 ± 3.1	10 ± 1.0
	150	109 ± 13.8	12 ± 2.1	23 ± 1.0	19 ± 1.7	11 ± 4.6
+	500	85 ± 10.1	11 ± 1.0	23 ± 0.6	18 ± 1.0	8 ± 0.6
+	1500	69 ± 3.2^P	6 ± 2.1^#	21 ± 1.0	17 ± 1.0	8 ± 0.6
+	5000	47 ± 15.3^P	0 ± 0.0^P,#	13 ± 2.1^P	7 ± 3.5^P	3 ± 3.5^P
Positive controls, +S9	Name	2-AA	2-AA	2-AA	B[a]P	2-AA
	Concentration [μg/plate]	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	118	15	29	20	13

DMSO: dimethylsulphoxide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

ENNG: N-ethyl-N-nitro-N-nitrosoguanidine

4-NQO: 4-nitroquinoline-N-oxide

B[a]P: benzo(a)pyrene

n.d.: not determined

P: precipitate

#: partial absence of bacterial background lawn

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA1535, TA1537, TA98, TA100 and WP2 uvrA) tested with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
The reliable GLP compliant OECD Guideline study was chosen. This study is sufficient to fulfil the standard information requirements set out in Annex VII, 8.4 of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

Based on the available data, there is no indication that the substance induces genetic toxicity. Nevertheless, no final decision on classification for genetic toxicity according to Regulation (EC) 1272/2008 can be made, as no information on chromosomal aberration and mutagenicity in mammalian cells/in vivo is available.