4-(1,1-dimethylethyl)cyclohexyl acrylate

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:

2009-10-27 to 2009-11-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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:

4-(1,1-dimethylethyl)cyclohexyl acrylate

EC Number:

282-104-8

EC Name:

4-(1,1-dimethylethyl)cyclohexyl acrylate

Cas Number:

84100-23-2

Molecular formula:

C13H22O2

IUPAC Name:

4-tert-butylcyclohexyl prop-2-enoate

Details on test material:

- Substance type: organic
- Analytical purity: 94.1%
- Physical state: liquid, yellowish, clear
- Storage condition of test material: refrigerator, protected from light

Method

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:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

22 µg - 5600 µg/plate (standard plate test, all tester strains)
1 µg - 250 µg/plate (preincubation test: TA 1535, TA 1537)
2 µg - 500 µg/plate (preincubation test: TA 100, TA 98)
22 µg - 5600 µg/plate (preincubation test: E. coli)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test item in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2-AA

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix (TA98, TA100, TA1535, TA1537, WP2 uvr A)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

MNNG

Positive control substance:

other: N-methyl-N`-nitro-N-nitrosoguanidine

Remarks:

without S9 mix (TA1535, TA100)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

NOPD

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

without S9 mix (TA 98)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

AAC

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix (TA1537)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

4-NQO

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix (WP2 uvr A)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 -72 hours in the dark

SELECTION AGENT (mutation assays): not used

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: reduced background lawn, decrease in number of revertants and reduction in the titer

Evaluation criteria:

Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control items both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria was ≥ 10^8/mL

The test item is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test item is generally considered non-mutagenic in this test if:
-The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Raw data is listed under "any other information on results incl. tables"

Cytotoxicity / choice of top concentrations:

other: Salmonella strains only depending on the strain and test conditions from about 125 µg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Raw data is listed under "any other information on results incl. tables"

Cytotoxicity / choice of top concentrations:

other: A bacteriotoxic effect was observed with the Salmonella strains only depending on the strain and test conditions from about 125 µg/plate onward.

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:

other: Salmonella strains only depending on the strain and test conditions from about 125 µg/plate onward.

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:

other: Salmonella strains only depending on the strain and test conditions from about 125 µg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No
- Water solubility: No
- Precipitation: No test item precipitation was found with and without S9 mix.

RANGE-FINDING/SCREENING STUDIES: Not performed

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control items both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants, reduction in the titer) was observed in the standard plate test using the Salmonella strains depending on the strain and test conditions from about 250 μg/plate onward. In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the number of his+ revertants, reduction in the titer) was observed depending on the strain and test conditions using the Salmonella strains from about 125 μg/plate onward. With the tester strain E. coli WP2uvrA no bacteriotoxicity was observed in all experiments up to the highest required concentration.

Any other information on results incl. tables

Table 1: First standard plate test

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E.coli
	TA1535	TA1537	TA98	TA100	WP2 uvr A
	Results with S9 mix
Solvent control	18	10	37	128	48
Positive control	125	177	674	896	227
22	20	10	36	119	48
112	19	9	29	101	41
560	9 B	3 B	15 B	10 B	48
2800	0 B	0 B	0 B	0 B	39
5600	0 B	0 B	0 B	0 B	43
	Results without S9 mix
Solvent control	18	11	33	120	42
Positive control	864	460	738	861	895
22	17	12	31	112	47
112	16	8	23	92	44
560	8 B	3 B	14 B	34 B	43
2800	0 B	0 B	0 B	0 B	44
5600	0 B	0 B	0 B	0 B	36

B= reduced background lawn

Table 2: Second standard plate test with S. typhimurium strains

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and E.coli
	TA1535	TA1537	TA98	TA100
	Results with S9 mix
Solvent control	20	9	32	94
Positive control	150	174	726	864
31.3	21	9	22	90
62.5	17	8	27	86
125	18	8	34	89
250	12	7	22	87
500	8 B	3 B	10 B	23 B
	Results without S9 mix
Solvent control	21	8	33	90
Positive control	992	456	471	945
31.3	21	8	32	85
62.5	20	7	29	77
125	19	8	31	90
250	11	6	34	78
500	6 B	4 B	13 B	21 B

B= reduce background lawn

Table 3 preincubation test with TA1535, TA1537

Dose (µg/plate)	Mean number of revertant colonies/3 replicates
	TA1535	TA1537
	Results with S9 mix
Solvent control	18	8
Positive control	140	134
1	17	5
5	16	7
25	17	7
125	15	6
250	5 B	3 B
	Results without S9 mix
Solvent control	18	10
Positive control	573	380
1	19	7
5	19	10
25	16	8
125	11	6
250	7 B	1 B

B= reduced background lawn

Table 4 preincubation test with TA98, TA100

Dose (µg/plate)	Mean number of revertant colonies/3 replicates
	TA100	TA 98
	Results with S9 mix
Solvent control	93	30
Positive control	939	566
2	98	25
10	101	29
50	87	25
250	83	27
500	19 B	9 B
	Results without S9 mix
Solvent control	95	28
Positive control	1038	414
2	94	25
10	97	32
50	103	22
250	81	24
500	19 B	14 B

B = reduced background lawn

Table 5 preincubation test with WP2 uvr A

Dose (µg/plate)	Mean number of revertant colonies/3 replicates
	WP2 uvr A
	Results with S9 mix
Solvent control	33
Positive control	240
22	31
112	34
560	29
2800	34
5600	33
	Results without S9 mix
Solvent control	48
Positive control	872
22	45
112	45
560	47
2800	46
5600	51