4-(1,1-dimethylethyl)cyclohexyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay: negative (BASF, 2009)

HPRT: negative (BASF 2015)

MNT in vitro: negative (BASF 2015)

Link to relevant study records

Reference

Reference 1

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

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)

Deviations:

no

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

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.

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)

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)

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)

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)

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.

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

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

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

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

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.

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

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 induced with Phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Experiment IA + II: 4.2; 8.4; 16.9; 33.7; 67.4; 134.9; 269.8; 539.5; 1079.0; 2158.0 (with S9 mix/ 4h)
Experiment IB: 0.08; 0.16; 0.3; 0.6; 1.3; 2.5; 5.0; 10.0; 20.0; 4.0 (without S9 mix /4 h)
Experiment II: 0.03; 0.07; 0.13; 0.26; 0.53; 1.05; 2.1; 4.2; 8.4; 16.9 (without S9 mix/24h)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Griseofulvin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h or 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2000

DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell counts (RICC) + proliferation index (PI)

Evaluation criteria:

A test item can be classified as mutagenic if:
− The number of micronucleated cells exceeds both the value of the concurrent negative control and the range of the historical negative control data.
− A significant, dose-related and reproducible increase in the number of cells containing micronuclei is observed

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Summary of results

Exp.	Preparation interval	Test item concentration in µg/ml	Proliferation Index	RICC in %	Cytotoxicity in %	Micronucleated Cells*                   in %
Exposure period 4 hrs without S9 mix
IB	24hrs	Solvent control^1	2.74	100	0	1.20
		Positive control^2	2.42	19	81	7.45^S
		0.08	2.86	171	0^#	0.90
		0.16	2.83	89	11	0.65
		0.3	2.86	44	56	0.90
		1.3	2.62	16	84	0.85
		2.5	2.66	-20	n.a.	0.60
Exposure period 24 hrs without S9 mix
II	24 hrs	Solvent control^1	2.88	100	0	0.85
		Positive control^3	2.55	63	37	7.95^S
		0.26	2.93	80	20	1.20
		0.53	2.93	54	46	1.20
		1.05	2.92	66	34	1.45
		2.1	2.80	21	79	0.95
* The number of micronucleated cells was determined in a sample of 2000 cells #Not cytotoxic since the RICC is higher than the solvent control value SNumber of micronucleated cells statistically significantly higher than corresponding control values n.a. Not analysable, because the cell number of the solvent control or treated cultures was lower after treatment 1DMSO 0.5 % (v/v) 2Mitomycin C 0.1μg/mL 3Griseofulvin 8.0μg/mL
Exposure period 4 hrs with S9 mix
IA	24 hrs	Solvent control^1	2.31	100	0	0.60
		Positive control^2	1.64	0	100	17.20^S
		16.9	2.40	64	36	1.15
		33.7	2.26	32	38	0.60
		67.4	2.34	54	46	0.65
		134.9	2.20	29	71	0.65
		269.8	2.13	-52	n.a.
II	24 hrs	Solvent control^1	2.84	100	0	1.20
		Positive control^2	1.64	-15	n.a.	12.45^S
		33.7	2.78	61	39	0.90
		67.4**	2.74	84	16	1.45
		134.9**	2.63	75	25	1.58
		269.8**	2.38	34	66	1.43

* The number of micronucleated cells was determined in a sample of 2000 cells

** The number of micronucleated cells was determined in a sample of 4000 cells

SNumber of micronucleated cells statistically significantly higher than corresponding control values

n.a. Not analysable, because the cell number of the solvent control or treated cultures was lower after treatment

1DMSO 0.5 % (v/v)

2CPA 10.0μg/mL

3CPA 15.0 μg/mL

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver mix S9 (induced with Phenobarbital/β-naphthoflavone)

Test concentrations with justification for top dose:

Test 1: 0.27; 0.55; 1.1; 2.2; 4.4 µg/mL (without S9 mix); 35.0; 70.0; 140.0; 210.0; 280.0 µg/mL (with S9 mix)
Test 2: 2.0; 3.0; 4.0; 6.0; 8.0 µg/mL (without S9 mix); 105.0; 140.0; 210.0; 245.0; 280.0 µg/mL (with S9 mix)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine'; 11 µg/mL

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.
Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, Laromer TBCH is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames

In a reverse gene mutation assay in bacteria according to OECD 471 and EU method B. 13 (BASF, 2009), strains of S. typhimurium (TA98, TA100, TA1535, TA1537) and E. coli (WP2 uvr A) were exposed to the test substance in DMSO at concentration ranges listed down below. A standard plate test (SPT) and a preincubation test (PIT) both with and without metabolic activation were performed.

 

22 - 5600 μg/plate (SPT, all test strains)

1 - 250 μg/plate (PIT: TA 1535, TA 1537)

2 - 500 μg/plate (PIT: TA 100, TA 98)

22 - 5600 μg/plate (PIT: E. coli)

 

Three independent experiments were performed, two standard plate tests and one preincubation test. No precipitation of the test item was found with and without S9 mix. A bacteriotoxic effect was observed only with the Salmonella strains in both the standard plate and the preincubation test from about 125 µL/plate onwards. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

HPRT

In a study according to OECD 476, two independent experiments each with and without metabolic activation were performed to assess the potential of Laromer TBCH to induce gene mutations in mammalian cells (BASF 2015). Exposure time was 4h in all experiments. The test substance was applied up to 280 µg/mL in the presence of S9 and up to 8µg/ml without the addition of S9. Concentrations were limited by cytotoxic effects. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

MNT in vitro

In a study according to OECD 487, Chinese hamster V79 cells were exposed to Laromer TBCH and evaluated for the induction of micronuclei (BASF 2015). The maximum concentrations used were limited by the cytotoxicity of the test item. In the absence of a metabolic activation system, up to 2.5 µg/mL or 2.1 µg/mL could be used, if the cells were treated for 4h or 24h, respectively. In the presence of S9 mix, the cells were treated for 4h with up to 269.8 µg/mL. In each experimental group two parallel cultures were analysed. Per culture at least 1000 cells were evaluated for cytogenetic damage. Neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. Appropriate mutagens (MMC, Griseofulvin and CPA) were used as positive controls. They induced statistically significant increases in the number of cells with micronuclei.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The registered substance did not cause gene mutations in bacteria or mammalian cells, nor chromosomal damage. Consequently, there is no need for classification for mutagenicity under Regulation (EC) No 1272/2008, as amended for the eighteenth time in Regulation (EU) 2022/692.