1-cyclohexylethyl (E)-but-2-enoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and TA 102 with and without metabolic activation

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Nov - 21 Dec 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Verkehr Baden-Württemberg, Stuttgart, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Experiment 1
without metabolic activation
5, 15, 50, 150, 500, 1500 and 5000 µg/plate for TA98
15, 50, 150, 500 and 1500 µg/plate for TA100
5, 15, 50, 150 and 500 µg/plate for TA102 and TA1537
50, 150, 500, 1500 and 5000 µg/plate for TA1535

with metabolic activation
15, 50, 150, 500, 1500 and 5000 µg/plate for TA98
50, 150, 500, 1500 and 5000 µg/plate for TA100, TA1535 and TA1537
15, 50, 150, 500 and 1500 µg/plate for TA102

Experiment 2
without metabolic activation
15, 50, 150, 500 and 1500 µg/plate for TA98, TA102 and TA1535
5, 15, 50, 150 and 500 µg/plate for TA1537
50, 150, 500, 1500 and 5000 µg/plate for TA100

with metabolic activation
15, 50, 150, 500 and 1500 µg/plate for TA102
50, 150, 500, 1500 and 5000 µg/plate for TA98, TA100, TA1535 and TA1537

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene (2-AA)

Remarks:

+S9: 2-AA (0.8 µg/plate, TA98, TA100, TA1535, TA102; 1.7 µg/plate,TA1537); -S9: NaN3 (0.7 µg/plate, TA100, TA1535); 2-NF (2.5 µg/plate, TA98); 9-AA (50 µg/plate, TA1537); MMC (0.15 µg/plate, TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies and/or a diminution of the background

Statistics:

X²-test was used to estimate the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level. Mean values and standard deviation were calculated.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: -S9: at 5000 µg/plate; Exp. 2: -S9: at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +S9: at 5000 µg/plate; -S9: at 500 µg/plate; Exp. 2: -S9: at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: -S9: at 5000 µg/plate; Exp. 2: -S9: at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +S9: at 1500 µg/plate; -S9: at 500 µg/plate; Exp. 2:+S9: at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound on the plates was not observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test substance was bacteriotoxic towards the strains TA1537 at 500 µg/plate and TA98 and TA1535 at 5000 µg/plate in Experiment 1 and towards the strains TA1537 at 500 µg/plate and TA98, TA1535 and TA102 at 1500 µg/plate in Experiment 2 without metabolic activation. In the presence of metabolic activation, the test substance was bacteriotoxic towards the strains TA102 at 1500 µg/plate and TA1537 at 5000 µg/plate in Experiment 1 and towards the strain TA102 at 1500 µg/plate in Experiment 2.

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 ± SD)
		Base-pair substitution type			Frameshift type
		TA100	TA102	TA1535	TA98	TA1537
-	0	124 ± 18	306 ± 40	31 ± 4	20 ± 3	10 ± 3
-	0 (DMSO)	99 ± 3	261 ± 7	31 ± 5	18 ± 5	11 ± 3
-	5	-	293 ± 23	-	20 ± 6	11 ± 2
-	15	108 ± 7	319 ± 10	-	20 ± 5	8 ± 1
-	50	113 ± 8	245 ± 30	28 ± 9	18 ± 2	11 ± 4
-	150	114 ± 3	230 ± 17	28 ± 4	10 ± 4	8 ± 3
-	500	102 ± 10	207 ± 16	20 ± 2	10 ± 2	4 ± 2^T
-	1500	104 ± 11	-	17 ± 5	12 ± 2	-
-	5000	-	-	13 ± 7^T	7 ± 3^T	-
Positive controls, –S9	Name	NaN3	MMC	NaN3	2-NF	9-AA
	Concentrations [μg/plate]	0.7	0.15	0.7	2.5	50
	Mean No. of colonies/plate (average of 3 ± SD)	330 ± 18	1018 ± 30	1065 ± 37	258 ± 18	220 ± 33
+	0	124 ± 17	398 ± 13	17 ± 3	23 ± 5	15 ± 3
+	0 (DMSO)	124 ± 5	376 ± 9	6 ± 3	21 ± 2	19 ± 4
+	15	-	363 ± 7	-	18 ± 4	-
+	50	124 ± 10	375 ± 10	8 ± 3	17 ± 4	18 ± 3
+	150	116 ± 18	310 ± 23	12 ± 4	18 ± 3	16 ± 5
+	500	116 ± 8	315 ± 12	6 ± 3	20 ± 10	11 ± 4
+	1500	111 ± 16	291 ± 36^T	8 ± 2	20 ± 3	15 ± 2
+	5000	108 ± 17	-	8 ± 3	17 ± 4	8 ± 3^T
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations [μg/plate]	0.8	0.8	0.8	0.8	1.7
	Mean No. of colonies/plate (average of 3 ± SD)	679 ± 85	576 ± 29	176 ± 16	491 ± 62	236 ± 53

DMSO: dimethylsulphoxide

NaN3: sodium azide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

MMC: mitomycin C

^T: bacteriotoxic

 

Table 2. Test results of Experiment 2 (plate incorporation).

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type			Frameshift type
		TA100	TA102	TA1535	TA98	TA1537
-	0	115 ± 2	298 ± 14	33 ± 5	32 ± 9	10 ± 4
-	0 (DMSO)	106 ± 5	332 ± 14	37 ± 4	24 ± 2	12 ± 2
-	5	-	-	-	-	15 ± 7
-	15	-	325 ± 14	27 ± 5	25 ± 3	14 ± 2
-	50	113 ± 7	300 ± 16	33 ± 4	18 ± 3	9 ± 2
-	150	98 ± 18	273 ± 13	24 ± 4	12 ± 4	12 ± 2
-	500	72 ± 15	233 ± 29	19 ± 9	15 ± 3	8 ± 1^T
-	1500	75 ± 6	213 ± 2^T	15 ± 3^T	10 ± 5^T	-
-	5000	76 ± 6	-	-	-	-
Positive controls, –S9	Name	NaN3	MMC	NaN3	2-NF	9-AA
	Concentrations [μg/plate]	0.7	0.15	0.7	2.5	50
	Mean No. of colonies/plate (average of 3 ± SD)	317 ± 50	583 ± 17	748 ± 32	323 ± 23	324 ± 46
+	0	123 ± 11	340 ± 25	13 ± 3	36 ± 2	15 ± 2
+	0 (DMSO)	106 ± 12	320 ± 22	12 ± 2	28 ± 2	19 ± 4
+	15	-	331 ± 21	-	-	-
+	50	117 ± 6	321 ± 22	11 ± 4	29 ± 3	17 ± 3
+	150	119 ± 8	308 ± 19	15 ± 3	24 ± 6	17 ± 7
+	500	106 ± 4	289 ± 41	14 ± 2	29 ± 6	20 ± 6
+	1500	121 ± 6	262 ± 24^T	9 ± 5	32 ± 2	15 ± 3
+	5000	102 ± 8	-	9 ± 2	26 ± 7	22 ± 3
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations [μg/plate]	0.8	0.8	0.8	0.8	1.7
	Mean No. of colonies/plate (average of 3 ± SD)	735 ± 68	431 ± 21	192 ± 18	481 ± 27	331 ± 57

DMSO: dimethylsulphoxide

NaN3: sodium azide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

MMC: mitomycin C

^T: bacteriotoxic

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation.

Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2000). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation either up to 5000 µg/plate (TA 100) or up to cytotoxic concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2000). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation either up to 5000 µg/plate (TA 100) or up to cytotoxic concentrations.

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008.