1-methylheptyl 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:

01 June 2017 - 01 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

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:

1-methylheptyl acrylate

EC Number:

256-005-5

EC Name:

1-methylheptyl acrylate

Cas Number:

42928-85-8

Molecular formula:

C11H20O2

IUPAC Name:

octan-2-yl prop-2-enoate

Test material form:

liquid

Method

Target gene:

Histidine operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route

Test concentrations with justification for top dose:

The highest recommended dose level of 5000 µg/plate was achievable. Thus, the dose levels selected for the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.
Without S9 mix, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose levels equal or above 500 µg/plate in the TA 100 strain, equal or above 1000 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 102 strain.
With S9 mix, a moderate toxicity was noted at dose levels equal or above 2500 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 100 strain.

Therfore, the selection of the highest dose level for the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

Without S9 mix:

Selected dose levels were:
- 20.6, 61.7, 185.2, 555.6, 1667 and 5000 µg/plate for the TA 102 strain in the 1st experiment,
- 6.86, 20.6, 61.7, 185.2, 555.6 and 1667 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the 1st experiment,
- 31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 1535, TA 1537, TA 98 and TA 102 strains in the 2nd experiment,
- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 100 strain in the 2nd experiment.

With S9 mix:

The selected dose levels were:
- 20.6, 61.7, 185.2, 555.6, 1667 and 5000 µg/plate for the 5 strains in the 1st experiment,
- 25, 50, 100, 200, 400 and 800 µg/plate for the TA 102 strain in the 2nd experiment,
- 12.5, 25, 50, 100, 200 and 400 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the 2nd experiment.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide (DMSO).
- Justification for choice: the test item was soluble in the vehicle at 100 mg/mL.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

see Table 7.6.1/1

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

see Table 7.6.1/1

Positive control substance:

benzo(a)pyrene

other: 2-anthramine

Remarks:

With S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

DURATION
- Preincubation period: 60 minutes, 37°C (for 2nd experiment with S9 mix only).
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn

NUMBER OF REPLICATIONS:
Three plates/dose level.

Evaluation criteria:

Acceptance criteria:
Each main experiment was considered valid if the following criteria are fully met:
-> the mean number of revertants in the vehicle controls is consistent with the historical data, in each strain and test condition (see table 7.6.1/2),
-> at least five analyzable dose levels (i.e. including at least three non-cytotoxic dose levels) are obtained for each strain and test condition,
-> the mean number of revertants in the positive controls is higher than that of the vehicle controls (at least 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or at least 3-fold increase (for the TA 1535 and TA 1537 strains)).
When these criteria were not met for one strain or test condition, the corresponding results were invalidated and the experiment was repeated.

Criteria for assessing mutagenic potential:
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

->The test item was considered to have shown mutagenic activity in the study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains)
in the mean number of revertants compared with the vehicle controls was observed, in any strain, at any dose level,
- and/or a reproducible dose-response relationship was evidenced.

->The test item was considered to have shown no mutagenic activity in the study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains)
or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, was observed at any of the tested dose levels,
- nor any evidence of a dose-response relationship was noted.

Statistics:

no

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see tables 7.6.1/2 to 7.6.1/5

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the main experiment, no precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.

RANGE-FINDING STUDY:
- No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.
- Without S9 mix, a moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose levels equal or above to 500 µg/plate in the TA 100 strain, equal or above to 1000 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 102 strain.
- With S9 mix, a moderate toxicity was noted at dose levels equal or above to 2500 µg/plate in the TA 98 strain and at 5000 µg/plate in the TA 100 strain.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean revertant colony counts for the vehicle controls were within the current historical control range of the laboratory (see table 7.6.1/ 2 in Any Other Information on Materials and methods).

RESULTS OF CYTOTOXICITY and GENOTOXICITY:

Experiments without S9 mix

- A moderate to strong toxicity was noted at dose levels equal or above 250 µg/plate in the TA 1535, TA 1537, TA 98 and TA 102 strains and at dose levels equal or above 125 µg/plate in the TA 100 strain.
- Slight increases in the number of revertants were noted in the TA 1537 strain in the second experiment. Since these increases did not reach the positive threshold of 3-fold the vehicle control value, were not dose-related and since no similar results were observed in the first experiment, they were considered to be non-biologically relevant.
No other noteworthy increase in the number of revertants was observed in any other strains.

Experiments with S9 mix

- When using the direct plate incorporation method (i.e. first experiment), a moderate to strong toxicity was noted at 5000 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains. When using the pre-incubation method (i.e. second experiment), a moderate to strong toxicity was noted at dose levels equal or above 200 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and at 800 µg/plate in the TA 102 strain. In the TA 1537 and TA 100 strains, the strong toxicity induced at 400 µg/plate prevented the scoring of revertant colonies (presence of microcolonies).
- The test item did not induce any noteworthy increase in the number of revertants, in any strains and test conditions.

Any other information on results incl. tables

7.6.1/2: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in the first test (direct plate incorporation method)

Strain	Compound	Dose-level (µg/plate)	With or without S9 mix	Number of revertant per plate	Mean revertant colony count (SD)
TA1535	NAN3	1	-	537-496-546	526.3 (26.7)
	TEST ITEM	1667	-	7St-9St-4St	6.7 (2.5)
		555.6	-	7Mt-3Mt-4Mt	4.7 (2.1)
		185.2	-	10-8-9	9.0 (1.0)
		61.7	-	6-6-10	7.3 (2.3)
		20.6	-	7-4-6	5.7 (1.5)
		6.86	-	8-7-10	8.3 (1.5)
	DMSO		-	12-8-9	9.7 (2.1)
TA1537	9 AA	50	-	88-51-75	71.3 (18.8)
	TEST ITEM	1667	-	2St-2St-3St	2.3 (0.6)
		555.6	-	3Mt-4Mt-4Mt	3.7 (0.6)
		185.2	-	4-4-6	4.7 (1.2)
		61.7	-	3-4-3	3.3 (0.6)
		20.6	-	6-7-4	5.7 (1.5)
		6.86	-	7-3-9	6.3 (3.1)
	DMSO		-	9-4-9	7.3 (2.9)
TA 98	2 NF	0.5	-	107-112-86	101.7 (13.8)
	TEST ITEM	1667	-	11St-12St-13St	12 (1.0)
		555.6	-	11Mt-12Mt-10Mt	11 (1.0)
		185.2	-	11-19-12	14.0 (4.4)
		61.7	-	13-19-15	15.7 (3.1)
		20.6	-	21-18-21	20 (1.7)
		6.86	-	29-11-10	16.7 (10.7)
	DMSO		-	18-10-21	16.3 (5.7)
TA 100	NAN3	1	-	484-473-433	463.3 (26.8)
	TEST ITEM	1667	-	60St-48St-95St	67.7 (24.4)
		555.6	-	76St-87St-85St	82.7 (5.9)
		185.2	-	79Mt-78Mt-113Mt	90 (19.9)
		61.7	-	85-72-70	75.7 (8.1)
		20.6	-	73-77-81	77 (4.0)
		6.86	-	76-79-60	71.7 (10.2)
	DMSO		-	72-102-85	86.3 (15.0)
TA102	MMC	0.5	-	1869-1985-2004	1952.7 (73.1)
	TEST ITEM	5000	-	182St-177St-188St	182.3 (5.5)
		1667	-	184St-151St-149ST	161.3 (19.7)
		555.6	-	201St-179St-140St	173.3 (30.9)
		185.2	-	341-318-369	342.7 (25.5)
		61.7	-	362-348-321	343.7 (20.8)
		20.6	-	411-445-417	424.3 (18.1)
	DMSO		-	445-409-399	417.7 (24.2)

7.6.1/3: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation in the first test (direct plate incorporation method) 

Strain	Compound	Dose-level (µg/plate)	With or without S9 mix	Number of revertant per plate	Mean revertant colony count (SD)
TA1535	2AM	2	+	138 -153 -135	142.0 (9.6)
	TEST ITEM	5000	+	8Mt-16Mt-11Mt	11.7 (4.0)
		1667	+	6-8-11	8.3 (2.5)
		555.6	+	9-16-13	12.7 (3.5)
		185.2	+	12-6-13	10.3 (3.8)
		61.7	+	10-12-8	10.0 (2.0)
		20.6	+	4-7-8	6.3 (2.1)
	DMSO		+	11-10-11	10.7 (0.6)
TA1537	2AM	2	+	78-87-82	82.3 (4.5)
	TEST ITEM	5000	+	7Mt-4Mt-7Mt	6.0 (1.7)
		1667	+	4-3-7	4.7 (2.1)
		555.6	+	10-8-9	9.0 (1.0)
		185.2	+	8-6-10	8.0 (2.0)
		61.7	+	23-6-7	12.0 (9.5)
		20.6	+	6-8-8	7.3 (1.2)
	DMSO		+	9-8-11	9.3 (1.5)
TA 98	2 AM	2	+	817-981-888	895.3 (82.2)
	TEST ITEM	5000	+	12Mt-20Mt-9Mt	13.7 (5.7)
		1667	+	15-17-19	17 (2.0)
		555.6	+	21-17-22	20.0 (2.6)
		185.2	+	19-19-26	21.3 (4.0)
		61.7	+	12-21-23	18.7(5.9)
		20.6	+	19-22-25	22.0 (3.0)
	DMSO		+	17-18-23	19.3 (3.2)
TA 100	BaP	5	+	1083-1085-1082	1083.3 (1.5)
	TEST ITEM	5000	+	85St-87St-104St	92.0 (10.4)
		1667	+	98-100-114	104.0 (8.7)
		555.6	+	108-104-103	105.0 (2.6)
		185.2	+	105-125-107	112.3 (11.0)
		61.7	+	102-124-108	111.3 (11.4)
		20.6	+	103-111-105	106.3 (4.2)
	DMSO		+	113-143-98	118.0 (22.9)
TA102	2AM	20	+	2633-2639-3372	2881.3 (424.9)
	TEST ITEM	5000	+	436-361-351	382.7 (46.5)
		1667	+	503-437-457	465.7(33.8)
		555.6	+	464-391-433	429.3 (36.6)
		185.2	+	448-479-423	450.0 (28.1)
		61.7	+	447-463-443	451.0 (10.6)
		20.6	+	489-504-470	487.7 (17.0)
	DMSO		+	499-514-441	484.7 (38.6)

 

 

7.6.1/4: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in the second test (direct plate incorporation method)

 

Strain	Compound	Dose-level (µg/plate)	With or without S9 mix	Number of revertant per plate	Mean revertant colony count (SD)
TA1535	NAN3	1	-	636-666-690	664.0 (27.1)
	TEST ITEM	1000	-	15Mt-11Mt-9Mt	11.7 (3.1)
		500	-	9Mt-13Mt-4Mt	8.7 (4.5)
		250	-	8Mt-7Mt-9Mt	8.0 (1.0)
		125	-	13-16-2	10.3 (7.4)
		62.5	-	9-6-13	9.3 (3.5)
		31.3	-	11-8-11	10.0 (1.7)
	DMSO		-	4-7-15	8.7 (5.7)
TA1537	9AA	50	-	210-292-287	263.0 (46.0)
	TEST ITEM	1000	-	4Mt-4Mt-6Mt	4.7 (1.2)
		500	-	8Mt-3Mt-4Mt	5.0 (2.6)
		250	-	9Mt-7Mt-3Mt	6.3 (3.1)
		125	-	3-1-9	4.3 (4.2)
		62.5	-	7-3-6	5.3 (2.1)
		31.3	-	9-8-6	7.7 (1.5)
	DMSO		-	2-3-3	2.7 (0.6)
TA 98	2 NF	0.5	-	151-139-143	144.3 (6.1)
	TEST ITEM	1000	-	20Mt-12Mt-17Mt	16.3 (4.0)
		500	-	20Mt-15Mt-17Mt	17.3 (2.5)
		250	-	12Mt-17Mt-17Mt	15.3 (2.9)
		125	-	7-17-16	13.3 (5.5)
		62.5	-	12-12-18	14.0 (3.5)
		31.3	-	13-15-11	13.0 (2.0)
	DMSO		-	17-18-17	17.3 (0.6)
TA 100	NAN3	1	-	559-568-595	574.0 (18.7)
	TEST ITEM	500	-	100Mt-115Mt-89Mt	101.3 (13.1)
		250	-	79Mt-97Mt-104Mt	93.3 (12.9)
		125	-	93Mt-79Mt-93Mt	88.3 (8.1)
		62.5	-	91-78-107	92.0 (14.5)
		31.3	-	88-97-105	96.7 (8.5)
		15.6	-	101-77-69	82.3 (16.7)
	DMSO		-	96-83-78	85.7 (9.3)
TA102	MMC	0.5	-	2462-2453-2235	2383.3 (128.5)
	TEST ITEM	1000	-	237St-219St-172St	209.3 (33.6)
		500	-	283Mt-335Mt-309Mt	309.0 (26.0)
		250	-	278Mt-332Mt-344Mt	318.0 (35.2)
		125	-	323-325-296	314.7 (16.2)
		62.5	-	379-342-381	367.3 (22.0)
		31.3	-	385-353-373	370.3 (16.2)
	DMSO		-	343-339-357	346.3 (9.5)

 

7.6.1/5: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation in the second test (preincubation method) 

Strain	Compound	Dose-level (µg/plate)	With or without S9 mix	Number of revertant per plate	Mean revertant colony count (SD)
TA1535	2AM	2	+	187-154-163	168.0 (17.1)
	TEST ITEM	400	+	6st-13St-3St	7.3 (5.1)
		200	+	12Mt-11Mt-7Mt	10.0 (2.6)
		100	+	11-12-10	11.0 (1.0)
		50	+	10-10-18	12.7 (4.6)
		25	+	13-16-12	13.7 (2.1)
		12.5	+	15-13-9	12.3 (3.1)
	DMSO		+	17-15-11	14.3 (3.1)
TA1537	2AM	2	+	63-65-70	66.0 (3.6)
	TEST ITEM	400	+	StUpMc-StUpMc-StUpMc
		200	+	6St-6St-8St	6.7 (1.2)
		100	+	6-7-6	6.3 (0.6)
		50	+	8-7-13	9.3 (3.2)
		25	+	17-16-12	15.0 (2.6)
		12.5	+	10-10-8	9.3 (1.2)
	DMSO		+	12-15-7	11.3 (4.0)
TA 98	2 AM	2	+	151-139-143	144.3 (6.1)
	TEST ITEM	400	+	16St-9St-10St	11.7 (3.8)
		200	+	17st-29St-21St	22.3 (6.1)
		100	+	25-25-27	25.7 (1.2)
		50	+	27-31-27	28.3 (2.3)
		25	+	23-38-16	25.7(11.2)
		12.5	+	26-21-23	23.3 (2.5)
	DMSO		+	27-20-21	22.7 (3.8)
TA 100	BaP	5	+	647-858-816	773.7 (111.7)
	TEST ITEM	400	+	StUpMc-StUpMc-StUpMc
		200	+	104St-93St-107St	101.3 (7.4)
		100	+	129-141-124	131.3 (8.7)
		50	+	149-149-144	147.3 (2.9)
		25	+	160-130-143	144.3 (15.0)
		12.5	+	133-142-133	136.0 (5.2)
	DMSO		+	102-119-126	115.7 (12.3)
TA102	2AM	20	+	1629-1883-1477	1663.0 (205.1)
	TEST ITEM	800	+	155St-219St-176St	183.3 (32.6)
		400	+	310-358-329	332.3 (24.2)
		200	+	423-378-391	397.3 (23.2)
		100	+	397-369-424	396.7 (27.5)
		50	+	461-433-377	423.7 (42.8)
		25	+	328-448-451	409.0 (70.2)
	DMSO		+	423-388-436	415.7 (24.8)

 

 

Applicant's summary and conclusion

Conclusions:

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of the study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium. The study was performed according to OECD No. 471 guideline and in compliance with the principles of Good Laboratory Practice. A preliminary toxicity test was performed to define the dose levels of the test item, dissolved in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to six dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose levels for each strain and test condition. The study was therefore considered to be valid. The selection of the highest dose level for the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose levels.

 

Without S9 mix, the selected dose levels were:

- 20.6, 61.7, 185.2, 555.6, 1667 and 5000 µg/plate for the TA 102 strain in the first experiment,

- 6.86, 20.6, 61.7, 185.2, 555.6 and 1667 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the first experiment,

- 31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 1535, TA 1537, TA 98 and TA 102 strains in the second experiment,

- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 100 strain in the second experiment.

 

A moderate to strong toxicity was noted at dose levels equal or above 250 µg/plate in the TA 1535, TA 1537, TA 98 and TA 102 strains and equal or above 125 µg/plate in the TA 100 strain. No increase in the number of revertants which could be considered as biologically relevant was observed in any tested strains.

With S9 mix, the selected dose levels were:

- 20.6, 61.7, 185.2, 555.6, 1667 and 5000 µg/plate for the five strains in the first experiment,

- 25, 50, 100, 200, 400 and 800 µg/plate for the TA 102 strain in the second experiment,

- 12.5, 25, 50, 100, 200 and 400 µg/plate for the TA 1535, TA 1537, TA 98 and TA 100 strains in the second experiment.

When using the direct plate incorporation method (i.e. first experiment), a moderate to strong toxicity was noted at 5000 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains. When using the pre-incubation method (i.e. second experiment), a moderate to strong toxicity was noted at dose levels equal or above 200 µg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and at 800 µg/plate in the TA 102 strain. In the TA 1537 and TA 100 strains, the strong toxicity induced at 400 µg/plate prevented the scoring of revertant colonies.The test item did not induce any noteworthy increase in the number of revertants, in any strains and test conditions.

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.