1-Propanol, 3-[[3-[[[3-(acetyloxy)-2,2-dimethylpropylidene]amino]methyl]-3,5,5-trimethylcyclohexyl]imino]-2,2-dimethyl-, 1-acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-03-18 to 2014-04-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

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:

(3E)-3-[(3-{[(E)-[2,2-dimethyl-3-(prop-1-en-2-yloxy)propylidene]amino]methyl}-3,5,5-trimethylcyclohexyl)imino]-2,2-dimethylpropyl acetate

EC Number:

805-722-7

Cas Number:

1064082-81-0

Molecular formula:

C24H42N2O4

IUPAC Name:

(3E)-3-[(3-{[(E)-[2,2-dimethyl-3-(prop-1-en-2-yloxy)propylidene]amino]methyl}-3,5,5-trimethylcyclohexyl)imino]-2,2-dimethylpropyl acetate

Test material form:

liquid

Method

Target gene:

Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. Main DNA target GC.
Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: S.ty.mur. TA98,100,1537,1535 rfa (cell wall), uvrB (DNA-repair) mutation

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver induced by Phenobarbital (PB) and β-naphthoflavone (BNF)

Test concentrations with justification for top dose:

5000; 1581, 500, 158, 50 and 15.8 μg/plate
Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test).

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Test item is good soluble in DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

E.coli and all of Salmonella strains with S9-mix

Controls 2

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.coli WP2 uvrA without S9-mix

Controls 3

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Salmonella TA1537 without S9-mix

Controls 4

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Salmonella TA100 and TA1535 without S9-mix

Controls 5

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD)

Remarks:

Salmonella TA98 without S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation) and pre-incubation
The Experiment I (Initial Mutation Test) of the main study the plate incorporation method was used. In the Experiment II (Confirmatory Mutation Test) the pre-incubation method was applied and the concentrations examined were the same as investigated in the Experiment I.

DURATION
- Preincubation period: 20 min at 37 °C (bacterial culture and the S9 Mix or phosphate buffer)
- Exposure duration: 48 hours in the dark at 37 °C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth; Toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment.

Evaluation criteria:

Evaluation of Experimental Data

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least


Conditions for the Validity of the Test

The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 10E9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.

Statistics:

None

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 nor precipitates, but tested up to recommended limit concentrations

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:

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

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:

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

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:

In the Confirmatory Mutation Test, the absence of revertant growth and reduced background lawn development was obtained in S. typhimurium TA1537 at 5000 μg/plate (-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:

In the Confirmatory Mutation Test, the absence of revertant growth and reduced background lawn development was obtained in S. typhimurium TA1535 at 5000 μg/plate (-S9 Mix) and in S. typhimurium TA1535 at 1581 μg/plate (-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

RANGE-FINDING/SCREENING STUDIES:
Pre-Experiment for Toxicity

The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
In the toxicity test the concentrations examined were: 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate.
The obtained revertant colony numbers were slightly lower than the revertant colony numbers of the vehicle control in S. typhimurium TA100, in the whole examined concentration range of 5000-5 μg/plate, with addition of metabolic activation (+S9 Mix).
Slightly higher revertant colony counts were obtained in S. typhimurium TA98 at the concentrations of 50 μg/plate, with addition of metabolic activation (+S9 Mix) and in TA100 in the concentration range of 5000-5 μg/plate, without metabolic activation (-S9 Mix).

COMPARISON WITH HISTORICAL CONTROL DATA: The obtained changes, slightly lower or higher revertant counts remained in the corresponding historical control data and biological variability range of the applied test system.

Any other information on results incl. tables

Table 1: Summary of results of initial mutation test

 

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	13.7	0.84	23.7	1.09	87.3	1.10	135.3	1.24	5.7	0.74	10.7	1.03	8.7	1.00	7.0	0.91	25.3	1.01	25.7	0.94
DMSO Control	16.3	1.00	21.7	1.00	79.7	1.00	109.0	1.00	7.7	1.00	10.3	1.00	8.7	1.00	7.7	1.00	25.0	1.00	27.3	1.00
Ultrapure Water Control	–	–	–	–	90.3	1.00	–	–	7.0	1.00	–	–	–	–	–	–	30.0	1.00	–	–
5000	11.3	0.69	21.0	0.97	114.0	1.43	132.7	1.22	10.0	1.30	9.3	0.90	12.3	1.42	10.3	1.35	20.7	0.83	26.3	0.96
1581	13.7	0.84	19.3	0.89	102.7	1.29	124.0	1.14	7.3	0.96	12.3	1.19	7.3	0.85	9.7	1.26	25.3	1.01	28.0	1.02
500	15.3	0.94	20.0	0.92	88.0	1.10	122.0	1.12	8.7	1.13	11.3	1.10	8.0	0.92	9.0	1.17	21.7	0.87	29.0	1.06
158	13.0	0.80	22.3	1.03	85.0	1.07	117.0	1.07	6.0	0.78	9.0	0.87	7.0	0.81	7.0	0.91	25.7	1.03	33.0	1.21
50	11.3	0.69	21.0	0.97	90.0	1.13	120.7	1.11	7.3	0.96	9.0	0.87	9.0	1.04	6.3	0.83	23.0	0.92	29.7	1.09
15.8	13.3	0.82	22.0	1.02	87.7	1.10	114.0	1.05	6.3	0.83	10.3	1.00	8.7	1.00	8.7	1.13	27.7	1.11	26.0	0.95
NPD (4mg)	173.3	10.61	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	948.0	10.49	–	–	729.3	104.19	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	452.7	52.23	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	826.0	27.53	–	–
2AA (2mg)	–	–	1725.3	79.63	–	–	1856.0	17.03	–	–	209.3	20.26	–	–	175.0	22.83	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	284.3	10.40

 

Table 2: Summary of results of confirmatory mutation test

 

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	16.7	1.09	23.3	1.06	108.7	1.32	139.3	1.23	9.3	0.93	17.7	1.20	6.3	0.79	7.3	0.79	31.3	1.12	42.0	1.09
DMSO Control	15.3	1.00	22.0	1.00	82.3	1.00	113.7	1.00	10.0	1.00	14.7	1.00	8.0	1.00	9.3	1.00	28.0	1.00	38.7	1.00
Ultrapure Water Control	–	–	–	–	78.7	1.00	–	–	9.3	1.00	–	–	–	–	–	–	40.0	1.00	–	–
5000	5.3	0.35	14.3	0.65	44.7	0.54	100.0	0.88	0.0	0.00	12.7	0.86	0.0	0.00	11.3	1.21	23.3	0.83	29.7	0.77
1581	13.0	0.85	20.7	0.94	82.7	1.00	112.7	0.99	5.7	0.57	14.3	0.98	7.3	0.92	9.0	0.96	32.7	1.17	39.7	1.03
500	13.7	0.89	20.0	0.91	95.3	1.16	114.3	1.01	10.3	1.03	13.0	0.89	6.3	0.79	7.0	0.75	35.0	1.25	43.7	1.13
158	14.3	0.93	23.0	1.05	89.7	1.09	115.7	1.02	10.0	1.00	11.0	0.75	6.7	0.83	8.3	0.89	31.3	1.12	41.0	1.06
50	13.7	0.89	20.3	0.92	84.7	1.03	127.3	1.12	10.3	1.03	13.3	0.91	7.7	0.96	7.0	0.75	28.0	1.00	43.3	1.12
15.8	14.3	0.93	24.3	1.11	87.3	1.06	122.3	1.08	11.7	1.17	14.0	0.95	8.7	1.08	8.0	0.86	24.0	0.86	40.0	1.03
NPD (4mg)	178.0	11.61	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1230.0	15.64	–	–	497.3	53.29	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	365.0	45.63	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1096.0	27.40	–	–
2AA (2mg)	–	–	709.3	32.24	–	–	1310.7	11.53	–	–	120.3	8.20	–	–	111.7	11.96	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	261.7	6.77

 

Table 3: Historical control data for Revertants/Plate (for the period of 2008-2013)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	22.6	110.1	10.1	7.5	24.7
		SD	3.3	31.0	1.4	2.7	4.2
		Minimum	11	66	2	2	11
		Maximum	40	162	23	19	41
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	29.6	117.7	12.0	8.7	34.2
		SD	3.6	22.9	1.6	2.3	3.5
		Minimum	13	76	4	2	20
		Maximum	48	170	24	20	54
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	22.1	106.0	9.9	7.4	23.8
		SD	3.1	29.3	1.2	2.8	3.5
		Minimum	11	66	3	2	10
		Maximum	40	156	23	18	42
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	28.6	117.4	12.1	8.4	34.2
		SD	3.4	22.1	1.6	2.2	3.8
		Minimum	17	73	3	2	17
		Maximum	49	168	25	18	55
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	23.8	110.3	9.9	6.5	25.2
		SD	6.8	30.9	1.4	3.4	3.8
		Minimum	9	70	2	1	12
		Maximum	43	159	24	16	45
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	29.8	118.8	11.8	7.8	35.1
		SD	7.4	23.0	1.5	3.4	4.9
		Minimum	10	81	3	2	19
		Maximum	48	174	24	20	55
			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	199.6	904.8	792.5	457.5	700.3
		SD	35.1	107.9	106.0	111.8	58.9
		Minimum	165	477	332	110	341
		Maximum	248	1953	1278	1439	1236
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	199.6	1340.5	173.3	152.1	280.8
		SD	35.1	347.7	37.6	23.3	80.0
		Minimum	248	491	87	68	144
		Maximum	165	2869	603	465	520

Applicant's summary and conclusion

Conclusions:

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The Bacterial Reverse Mutation Assay (using Salmonella typhimurium and Escherichia coli) with the test item was conducted according to the OECD guideline 471 and GLP. The test item was suspended respectively dissolved in dimethyl sulfoxide (DMSO). Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential. in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate. The tested test item concentrations were: 5000, 1581, 500, 158, 50 and 15.8 μg/plate.

 

In the performed experiments positive and negative (vehicle) controls were run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls and were within the corresponding historical control data ranges. The reference mutagens showed a distinct increase of induced revertant colonies. In the performed experimental phases at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain were applied. The validity criteria of the study were fulfilled.

 

No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. Slight, unequivocal inhibitory effect of the test item was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in the examined Salmonella typhimurium strains at the concentration of 5000 μg/plate in absence of exogenous metabolic activation (-S9 Mix).

 

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.