Allyl (cyclohexyloxy)acetate

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:

24 Aug - 24 Sep 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

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

Test material

Test material information

Constituent 1

Reference substance name:

Allyl (cyclohexyloxy)acetate

EC Number:

272-657-3

EC Name:

Allyl (cyclohexyloxy)acetate

Cas Number:

68901-15-5

Molecular formula:

C11H18O3

IUPAC Name:

allyl (cyclohexyloxy)acetate

Method

Target gene:

his operon

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone/ß-naphthoflavone (80/100 mg/kg bw/d, oral)

Test concentrations with justification for top dose:

Preliminary toxicity study:
0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation for TA100

First experiment:
15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA98
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA100, TA1535, TA102 and TA1537
5, 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for TA1535
5, 15, 50, 150, 500 and 1500 µg/plate with metabolic activation for TA100, TA102, TA98 and TA1537

Second experiment:
15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for all strains
5, 15, 50, 150, 500 and 1500 µg/plate with metabolic activation for all strains

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

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

mitomycin C

other: 2-Aminoanthracene (2AA); 1,8- dihydroxyanthraquinon (DANTHRON)

Details on test system and experimental conditions:

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

DURATION
- Exposure duration: approx. 48 h

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the bacterial background lawn and/or a reduction in the frequency of revertant colonies

Evaluation criteria:

The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

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:

starting at 500 µg/plate with and without S9-mix

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 500 µg/plate with and without S9-mix

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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 500 µg/plate with and without S9-mix

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 500 µg/plate with and without S9-mix

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:

cytotoxicity

Remarks:

starting at 500 µg/plate with and without S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material. The test material was toxic at 5000 µg/plate to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be sterile.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the bacterial background lawn and/or a reduction in the frequency of revertant colonies in all of the tester strains both with and without metabolic activation. The first evidence of a weakened bacterial background lawn was observed at 500 µg/plate. Generally, the test material induced a greater toxic response after the addition of S9-mix. The sensitivity of the bacterial strains to the toxicity of the test material varied between strains and between exposures with or without S9-mix. The test material was, therefore, tested up to its toxic limit.

Any other information on results incl. tables

Table 1. Test results of experiment 1

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	TA102	TA98	TA1537
–	0	128	20	247	24	7
–	0 (DMSO)	114 ± 13.7	30 ± 3.5	317 ± 36.4	28 ± 3.5	13 ± 2.3
–	15	NT	NT	NT	25 ± 4.0	NT
–	50	109 ± 4.4	19 ± 5.5	278 ± 3.6	22 ± 3.2	11 ± 2.9
–	150	112 ± 12.0	20 ± 2.6	281 ± 31.8	31 ± 6.5	12 ± 4.5
–	500	105 ± 2.1	15 ± 5.5	279 ± 22.2	28 ± 2.0	11 ± 1.0
–	1500	67 ± 6.1	5 ± 1.7	107 ± 12.1	10 ± 2.9	5 ± 4.7
–	5000	0	0	0^T	0	0
Positive controls, –S9	Name	ENNG	ENNG	MMC	4-NQO	9-AA
	Concentrations (μg/plate)	3	5	0.5	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	539 ± 64.2	222 ± 39.2	751 ± 184.5	171 ± 19.0	784 ± 250.9
+	0	143	22	310	-	10
+	0 (DMSO)	145 ± 3.0	12 ± 3.5	310 ± 28.7	39 ± 1.7	16 ± 2.6
+	5	119 ± 9.8	12 ± 0.6	284 ± 63.4	40 ± 3.1	17 ± 7.8
+	15	118 ± 6.4	17 ± 4.5	318 ± 51.8	27 ± 8.1	15 ± 6.7
+	50	120 ± 14.7	10 ± 1.7	333 ± 21.7	29 ± 2.5	9 ± 2.1
+	150	98 ± 4.7	13 ± 2.6	296 ± 5.8	31 ± 4.0	10 ± 2.0
+	500	85 ± 6.1	6 ± 2.6	0^V	12 ± 3.8	0^V
+	1500	0^V	0^V	0^V	0^V	0^T
+	5000	NT	0^T	NT	NT	NT
Positive controls, +S9	Name	2-AA	2-AA	DAN	B[a]P	2-AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1770 ± 182.4	192 ± 16.2	707 ± 65.6	255 ± 6.7	685 ± 40.8

DMSO: Dimethylsulphoxide

NT: Not tested at this dose level

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

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

4-NQO: 4-nitroquinoline-N-oxide

MMC: Mitomycin C

B[a]P: Benzo(a)pyrene

DAN: 1,8-dihydroxyanthraquinone

T: Toxic

V: Very weak lawn

Table 2. Test results of experiment 2.

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	TA102	TA98	TA1537
–	0	145	20	306	32	12
–	0 (DMSO)	147 ± 5.7	20 ± 1.5	339 ± 3.2	33 ± 4.0	12 ± 5.9
–	15	127 ± 17.7	18 ± 0.6	295 ± 23.0	38 ± 6.5	12 ± 4.9
–	50	146 ± 7.6	18 ± 2.6	300 ± 16.1	28 ± 5.3	5 ± 1.7
–	150	124 ± 4.5	15 ± 0.6	297 ± 29.4	22 ± 5.0	9 ± 4.6
–	500	110 ± 26.5	10 ± 0.6	310 ± 14.9	22 ± 5.5	6 ± 3.1
–	1500	63 ± 5.6	4 ± 2.1	158 ± 18.7	9 ± 3.5	6 ± 3.6
–	5000	0	2 ± 1.5	0	1 ± 1.2	4 ± 3.2^S
Positive controls, –S9	Name	ENNG	ENNG	MMC	4-NQO	9-AA
	Concentrations (μg/plate)	3	5	0.5	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	471 ± 47.3	144 ± 20.9	1054 ± 39.6	139 ± 52.3	1032 ± 104.8
+	0	90	-	-	-	-
+	0 (DMSO)	107 ± 9.5	17 ± 5.0	302 ± 17.1	32 ± 3.5	10 ± 2.3
+	5	103 ± 3.8	15 ± 0.6	302 ± 34.8	28 ± 2.6	10 ± 4.4
+	15	100 ± 4.6	11 ± 2.9	310 ± 9.2	28 ± 4.6	8 ± 0.6
+	50	88 ± 20.3	16 ± 5.7	289 ± 12.9	38 ± 7.1	8 ± 0.6
+	150	85 ± 7.6	15 ± 4.2	265 ± 11.1	29 ± 1.5	3 ± 1.0
+	500	59 ± 2.5	11 ± 0.6	0^S	10 ± 7.4^S	0^V
+	1500	21 ± 4.0^V	2 ± 2.1^S	0^V	9 ± 3.1^S	0^T
Positive controls, +S9	Name	2-AA	2-AA	DAN	B[a]P	2-AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1277 ± 77.7	124 ± 7.4	727 ± 165.4	188 ± 13.2	169 ± 12.0

DMSO: Dimethylsulphoxide

NT: Not tested at this dose level

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

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

4-NQO: 4-nitroquinoline-N-oxide

MMC: Mitomycin C

B[a]P: Benzo(a)pyrene

DAN: 1,8-dihydroxyanthraquinone

S: Sparse lawn

T: Toxic

V: Very weak lawn

Table 3. Historical control data

positive control values up to and including 1997
Strain	TA100		TA98		TA1535		TA1538		TA1537		WP2uvrA		TA102
S9-Mix	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
Mean	117	120	28	33	24	18	21	25	10	11	20	21	264	283
SD	25	22	7	7	5	4	7	6	2	2	5	s	30	33
Min	61	63	13	14	10	11	6	11	4	5	12	11	216	205
Max	196	177	56	52	39	39	41	50	17.	20	.39	40	339	343
Values	1603	863	1047	837	948	718	309	190	938	703	634	436	197	100
vehicle control values up to and including 1997
Strain	TA100		TA98		TA1535		TA1538		TA1537		WP2uvrA		TA102
S9-Mix	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
Mean	613	949	203	420	466	291	484	454	812	266	834	734	785	705
SD	176	190	57	113	208	64	146	131	201	90	197	211	172	127
Min	277	412	127	198	163	139	190	212	161	123	342	225	540	511
M ax	1126	1315	698	757	1005	516	799	915	1149	718	1209	1089	1188	1090
Values	195	195	194	195	180	181	93	92	182	183	162	157	76	72
positive control values up to and including 1998
Strain	TA100		TA98		TA1535		TA1538		TA1537		WP2uvrA		TA102
S9-Mix	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
Mean	124	125	31	36	26	18	26	28	11	14	23	25	262	285
SD	16	16	6	6	5	4	6	5	3	3	4	4	31	32
Min	73	81	15	22	12	10	15	18	5	6	13	13	191	231
Max	173	170	45	54	38	37	36	36	20	23	34	41	304	363
Values	342	339	337	340	341	331	16	16	323	325	287	285	20	22
vehicle control values up to and including 1998
Strain	TA100		TA98		TA1535		TA1538		TA1537		WP2uvrA		TA102
S9-Mix	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
Mean	556	1209	143	472	375	260	403	556	1052	298	851	837	863	747
SD	114	318	24	119	173	64	93	197	259	106	202	223	127	116
Min	349	681	101	198	112	105	305	344	173	123	415	260	646	591
Max	1147	2740	225	733	1143	601	604	1073	1912	652	1486	1380	1096	1096
Values	141	141	140	143	142	141	12	12	142	141	138	138	24	25

SD = standard deviation

Min = minimal value

Max = maximal value

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA1535, TA1537, TA98, TA100 and TA102) with and without metabolic activation tested.