Anisole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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:

From 23 April 2012 to 13 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 471 guideline study in compliance with the GLP. No deviation from the protocol of the study.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other 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:

not specified

Qualifier:

according to guideline

Guideline:

other: EPA, OPPTS 870.5100 (Public Draft, June 1996)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2(R1), 09 November 2011

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine: TA 1535, TA 100, TA 1537 and TA 98
-Tryptophan: Escherichia coli WP2 uvr A

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: see Table 7.6.1/1

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: see Table 7.6.1/1

Metabolic activation:

with and without

Metabolic activation system:

S9 from induced phenobarbital/Beta-naphthoflavone rat liver.

Test concentrations with justification for top dose:

- Range finding test: 10; 31.6; 100; 316; 1000; 2500 and 5000 µg/plate
- Initial mutation test (plate incorporation method) and confirmatory mutation test (pre-incubation method): 5; 15.81; 50; 158.1; 500, 1581 and
5000 µg/plate
- Complementary confirmatory mutation test: 0.5; 15.81; 50; 158.1; 500 and 1581 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (test item) and Water (positive control)
- Justification for choice of solvent/vehicle: in this study, two vehicle control groups were used depending on the solubility of the test item and the solubility of strain specific positive chemicals. The test item was partially insoluble in Distilled water. Therefore, the solubility of the test item was examined in Dimethyl sulfoxide (DMSO) and Acetone. The test item was soluble in the two examined solvents. Due to the better biocompatibility to the test system, DMSO was selected for solvent of the study.

Untreated negative controls:

yes

Remarks:

(untreated)

Negative solvent / vehicle controls:

yes

Remarks:

(two vehicle control groups: DMSO and water)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

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

Remarks:

see Table 7.6.1/2

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- In agar (plate incorporation): In the Range Finding Test as well as in the Initial Mutation Test
- preincubation: in the Confirmatory Mutation Test and complementary mutation test

DURATION
- Incubation period: at 37°C for 48 hours.
- Preincubation period: 20 min at 37ºC

NUMBER OF REPLICATIONS: 3 plates/dose/strain. Two independent experiments were performed

DETERMINATION OF CYTOTOXICITY
- Method: the cytotoxicity of the test material was determined using TA100 and TA98 in the presence and absence of metabolic activation system (+/-S9 Mix) with appropriate untreated, negative (solvent) and positive controls. In the test each samples (including the controls) were tested in triplicate. The concentrations examined were 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate. After 48 hours incubation at 37°C, the plates were scored for revertant colonies and examined for a thinning of the background lawn.

Evaluation criteria:

The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

- Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

Criteria for a Negative Response:
A test article was considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

No data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See below for more details

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See below for more details

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: the range finding was performed using Salmonella typhimurium TA 98 and TA 100 strains. The concentrations examined were 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate. Slightly higher numbers of revertant colonies compared to the solvent control plates were observed in TA 98 strain without S9 mix. However, they had no biological significance and were considered as reflecting the variability of the test system. Slightly lower numbers of revertant colonies compared to the DMSO solvent control plates were observed in TA 98 strain with S9 mix at some non-cytotoxic concentrations. However, the observed revertant counts were within the historical control range and without biological significance in all cases. Slight inhibitory, cytotoxic effect of the test item (slightly reduced background lawn) was observed in the preliminary experiment in both examined strains at 5000 µg/plate with S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: the number of revertants colonies was within the historical control range (the initial mutation test, confirmatory mutation test, vehicle and positive controls)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the confirmatory mutation test, excessive cytotoxicity was observed in S.typhimurium TA100 and TA1535 strains without metabolic activation at the three highest concentrations. In these cases, the number of analyzable doses did not meet the recommandations of the test guidelines. Therefore, an additional experiment (complementary confirmatory mutation test) will be performed in these strains in an additional experimental period to complete the data. The experimental conditions were the same as in the confirmatory mutation test.

Slight inhibitory, cytotoxic effect of the test item was observed in the initial mutation test in S. typhimurium TA98 and TA100 strains at 5000 µg/plate with S9 mix. Similar, but stronger effect was observed in the confirmatory mutation test in S. typhimurium TA98 and TA1537 and Escherichia coli WP2 uvrA strains at 5000 and 1581 µg/plate without S9 mix, and in all the five tester strains at 5000 and 1581 µg/plate with S9 mix, and in the complementary confirmatory mutation test in S. typhimurium TA100 and TA1535 strains at 1581 and 500 µg/plate without S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/3: Summary Table of the Initial Mutation Test 

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	30.0	40.0	91.7	101.7	8.0	11.0	6.3	7.3	23.3	43.3
	MF	1.02	1.32	1.01	0.98	1.09	1.27	1.12	0.85	1.17	1.15
DMSO control	Mean	29.3	30.3	90.7	104.0	7.3	8.7	5.7	8.7	20.0	37.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
Distilled water control	Mean	--	--	94.7	--	8.0	--	--	--	29.0	--
	MF	--	--	1.04	--	1.09	--	--	--	1.45	--
5000	Mean	31.3	30.3	67.7	92.0	6.3	6.7	4.7	6.7	26.7	27.3
	MF	1.07	1.00	0.75	0.88	0.86	0.77	0.82	0.77	1.33	0.73
1581	Mean	30.0	36.0	77.7	96.7	10.0	10.0	5.0	6.7	26.0	34.3
	MF	1.02	1.19	0.86	0.93	1.36	1.15	0.88	0.77	1.30	0.91
500	Mean	31.0	43.0	102.0	102.7	9.0	10.7	5.3	7.7	28.3	36.7
	MF	1.06	1.42	1.13	0.99	1.23	1.23	0.94	0.88	1.42	0.97
158.1	Mean	34.7	40.0	94.0	120.0	10.7	12.0	3.0	7.0	35.3	32.7
	MF	1.18	1.32	1.04	1.15	1.45	1.38	0.53	0.81	1.77	0.87
50	Mean	30.7	37.7	104.3	124.0	8.0	14.7	7.7	6.3	31.0	39.7
	MF	1.05	1.24	1.15	1.19	1.09	1.69	1.35	0.73	1.55	1.05
15.81	Mean	28.7	38.7	99.0	111.7	10.3	12.3	3.7	8.3	32.3	37.0
	MF	0.98	1.27	1.09	1.07	1.41	1.42	0.65	0.96	1.62	0.98
5	Mean	26.0	42.7	92.7	113.7	9.0	10.0	5.3	6.3	23.7	40.0
	MF	0.89	1.41	1.02	1.09	1.23	1.15	0.94	0.73	1.18	1.06
NPD (4µg)	Mean	353.3	--	--	--	--	--	--	--	--	--
	MF	12.05	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2370.7	--	2422.7	--	202.3	--	211.0	--	--
	MF	--	78.15	--	23.29	--	23.35	--	24.35	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	301.7
	MF	--	--	--	--	--	--	--	--	--	8.01
SAZ (2µg)	Mean	--	--	1337.3	--	1172.0	--	--	--	--	--
	MF	--	--	14.13	--	146.50	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	521.3	--	--	--
	MF	--	--	--	--	--	--	92.00	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1110.7	--
	MF	--	--	--	--	--	--	--	--	38.30	--

Notes: NPD: 4-nitro-1,2-phenylene-diamine; 2AA: 2-aminoanthracene; SAZ: sodium azide; 9AA: 9-aminoacridine; MMS: Methyl-methanesulfonate.  

Conclusions:

Interpretation of results:
negative (with and without metabolic activation)

Under the test conditions of this study, Anisole had no mutagenic activity in the applied bacterium tester strains.

Executive summary:

In a reverse gene mutation assay in bacteria (Hargitai, 2012) performed according to the OECD N° 471 guideline and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were exposed to the test item Anisole diluted in DMSO at concentrations ranging from 0.5 to 5000 µg/plate in the presence and absence of metabolic activation system from liver fraction of phenobarbital/beta-naphthoflavone-induced rats (S9-mix).

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Pre-Incubation Method) and a Complementary Confirmatory Mutation Test (Pre-Incubation Method) because of excessive cytotoxicity observed in the confirmatory mutation test for some conditions.

 Based on the results of the range finding test, the test item concentrations in the initial mutation test and confirmatory mutation test were 5000; 1581; 500; 158.1; 50, 15.81 and 5µg/plate. Based on the results of the confirmatory mutation test, the test item concentrations in the complementary confirmatory mutation test were 1581; 500; 158.1; 50, 15.81; 5; 1.581 and 0.5 µg/plate.

 

In the initial mutation test, confirmatory mutation test and complementary confirmatory mutation test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies were below the biological relevance when compared with the solvent controls, within the historical control range and within the normal biological variability of the test system.

Slight inhibitory, cytotoxic effect of the test item was observed in the initial mutation test TA98 and TA100 strains at 5000 µg/plate with metabolic activation. Similar, but stronger effect was observed using the pre-incubation method, in the confirmatory mutation test in TA98, TA1537 andE.coli WP2uvrA strains at 5000 and 1581 µg/plate without metabolic activation, and in all the five tester strains at 5000 and 1581 µg/plate with metabolic activation, and in the complementary confirmatory mutation test in TA100 and TA1535 strains at 5000 and 1581 µg/plate without metabolic activation.

The positive controls induced the appropriate responses in the corresponding strains.

Under the test conditions, Anisole did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used with and without metabolic activation during the study.

In conclusion, Anisole had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

This study is considered as acceptable as it satisfied the criteria of the OECD guideline N° 471.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 June, 2012 to 10 October, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-conducted study, according to guideline, GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2 (R1), "Guidance on Genotoxicity Testing And Data Interpretation for Pharmaceuticals Intended For Human Use", 09 November 2011.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

tk locus (TK+/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium + Antibiotic-antimycotic solution + Pluronic-F68 + Pyruvic acid + NaHCO3
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix was prepared from liver of a group of Wistar male rats induced by Phenobarbital/Beta-Naphthoflavone. The S9 fraction was dispensed into aliquots at -80 +/- 10°C until required.

Test concentrations with justification for top dose:

- In the range finding pre-experiment: 0.25, 0.76, 2.29, 6.86, 20.58, 61.73, 185.2, 555.6, 1666.7, 5000 µg/mL ( +/- S9 mix)
- In main experiments (exp I ): 31.25, 62.5, 125, 250, 500, 750, 875, 1000, 1125, 1250, 1500, 1750 and 2000 µg/mL (+/- S9 mix)
- In main experiments (exp II): 31.25, 62.5, 125, 250, 500, 750, 875, 1000, 1125, 1250, 1500, 1750 and 2000 µg/mL (+ S9 mix)
- In main experiments (exp II): 31.25, 62.5, 125, 250, 375, 500, 700, 900, 1100, 1300, 1500, 1700 and 2000 µg/mL (- S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen according to its solubility properties.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. Two independent experiments (a first experiment as preliminary range-finding test and a second test as main experiments (experiment I + experiment II) according to the metabolic activation method (with and without S9 mix).

DURATION
- Exposure duration:
* Range finding pre-experiment: 3 h (+ S9), 3 h and 24 h (- S9)
* Main study, experiment I: 3 h (+/- S9)
* Main study, experiment II, 3 h (+ S9), and 24 h (- S9)
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): two weeks
- Fixation time (start of exposure up to fixation or harvest of cells): seveteen days

SELECTION AGENT (mutation assays):
after expression time, the cell concentration was adjusted to 1X10000 cells/mL. TFT (300 µg/mL stock solution) was diluted 100-fold into these
suspensions to give a final concentration of 3 µg/mL. 0.2 mL of each suspension was placed into each well of four, 96-well microplates at 2000 cells per well. Microplates were incubated at 37°C +/- 0.5°C containing approximately 5% (v/v) CO2 in air for approximately two weeks.

NUMBER OF REPLICATIONS: duplicate cultures for negative control, positive control and test substance dose levels.

NUMBER OF CELLS EVALUATED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Other: large and small colonies were scored.
OTHER:
- The pH and the osmolarity value were determined in culture medium

Evaluation criteria:

The test item was considered to be mutagenic in this assay if all the following criteria were met:
- the assay is valid
- statistically significant (p<0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the coresponding negative control values at one or more concentrations
- the increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions)
- there is a significant concentration-relationship as indicated by the linear trend analysis (p<0.05)
- the mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 1000000 viable cells (GEF: the Global Evaluation Factor) higher than the corresponding negative control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies (gotal wells with clones) was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity to obtain a modified estimate of variance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1125 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there were no large changes in pH after treatment in both experiments.
- Effects of osmolality: there were no large changes in osmolality after treatment in both experiments.
- Evaporation from medium: no data
- Precipitation: no precipitation was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
The test item was examined up to 5000 µg/mL in the Preliminary Toxicity Test. No insolubility, but cytotoxicity was observed, therefore the concentration selected for the main experiments were expected to cover the concentration range from the maximum cytotoxicity (resulting approximately
10-20% relative survival) to little or no cytotoxicity. The selected highest concentration was 2000 µg/mL. At least thirteen test concentrations were selected for the main experiments.

COMPARISON WITH HISTORICAL CONTROL DATA:
Solvent and positive controls are in the range of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Assay 1, following a 3-hour treatment with metabolic activation, expressive cytotoxicity of the test item was observed at 2000, 1750, 1500, 1250 and 1125 µg/mL concentrations, cell in these samples died during the treatment or in the expression period. An evaluation was made using data of first surviving concentration of 1000 µg/mL (relative survival value of 17 %).

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at 2000, 1750, 1500, 1250 and 1125 µg/mL concentrations, cells in these samples died during the treatment or in the expression period. Therefore, an evaluation was made using data of the first surviving concentration of 1000 µg/mL (relative survival value of 28%) and the next seven concentrations (a total of eight samples).

In Assay 2, following a 3-hour treatment with metabolic activation, similarly to the first test, excessive cytotoxicity of the test item was observed at 2000 and 1750 µg/mL concentrations, cells in these samples died during the treatment or in the expression period. Marked cytotoxicity of the test item was still detected at 1500, 1250 and 1125 µg/mL concentrations (relative survival values of 4, 6 and 8 %, respectively), therefore these concentrations were excluded from the evaluation. An evaluation was made using data of the next concentration of 1000 µg/mL (relative survival value of 18 %) and seven lower concentrations (a total of eight samples).

In Assay 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at 2000, 1700, 1500, 1300 and 1100 µg/mL concentrations, cells in these samples died during the treatment. The relative survival value of the first surviving concentration of 900 µg/mL was 10 %. Therefore, an evaluation was made using data of all examined concentrations from 900 µg/mL to 31.25 µg/mL (a total of eight samples)

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/1: Mutagenicity Results of Assay 1 (3 hour treatment in the presence of S9-mix)

S9 mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn^2/var(Dn)¨	Mutation frequency
+	3	2000 μg/mL	ND	ND	ND	ND	ND
		1750 μg/mL	ND	ND	ND	ND	ND
		1500 μg/mL	ND	ND	ND	ND	ND
		1250 μg/mL	ND	ND	ND	ND	ND
		1125 μg/mL	ND	ND	ND	ND	ND
		1000 μg/mL	641/768	54/768	73/768	0.297	110.7
		875 μg/mL	637/768	63/768	68/768	2.488	144.1
		750 μg/mL	668/768	48/768	52/768	0.294	111.4
		500 μg/mL	655/768	52/768	61/768	0.001	95.3
		250 μg/mL	666/768	62/768	40/768	0.628	78.0
		125 μg/mL	665/768	43/768	60/768	0.007	94.2
		62.5 μg/mL	689/768	29/768	50/768	0.389	80.7
		31.25 μg/mL	664/768	59/768	45/768	0.055	90.4
		Solvent control	660/768	49/768	59/768	--	96.3
		Untreated control	700/768	25/768	43/768	--	63.3
		Positive control (CP: 4 μg/mL)	308/768	117/768	343/768	¨¨ 8.15E-11	1023.5*

 

In linear trend analysis β²/var (β) = 3.67, significant.

 

* = Statistically significant.

¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) >5.66(at p<0.05).

¨¨= Evaluated by T-test for independent samples. Significant at p<0.05.

 

D_n= Difference of log mutant frequency of dose “n” and that of the Vehicle control

var(D_n) = variance of D_n                              β = slope of the curve                    var(β) = variance of the slopE

Solvent control = 1 (v/v) % Dimethyl sulfoxide

 CP = Cyclophosphamide

ND = No data (No cells were plated for colony growing due to excessive cytotoxicity observed during the treatment or in the expression period.)

 

Table 7.6.1/2: Mutagenicity Results of Assay 1 (3 hour treatment in the absence of S9-mix) 

S9 mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn^2/var(Dn)¨	Mutation frequency
-	3	2000 μg/mL	ND	ND	ND	ND	ND
		1750 μg/mL	ND	ND	ND	ND	ND
		1500 μg/mL	ND	ND	ND	ND	ND
		1250 μg/mL	ND	ND	ND	ND	ND
		1125 μg/mL	ND	ND	ND	ND	ND
		1000 μg/mL	682/768	39/768	47/768	0.816	85.9
		875 μg/mL	671/768	47/768	50/768	0.686	88.3
		750 μg/mL	639/768	69/768	60/768	0.034	106.1
		500 μg/mL	647/768	66/768	55/768	0.884	143.9
		250 μg/mL	680/768	33/768	55/768	0.017	107.3
		125 μg/mL	682/768	45/768	41/768	0.131	100.4
		62.5 μg/mL	681/768	48/768	39/768	0.879	85.1
		31.25 μg/mL	667/768	47/768	54/768	0.102	101.9
		Solvent control	659/768	54/768	55/768	--	111.5
		Untreated control	670/768	53/768	45/768	--	77.6
		Positive control (NQO: 0.15 μg/mL)	405/768	145/768	218/768	¨¨ 6.40E-10	628.3*

In linear trend analysis β²/var (β) = 0.25, not significant.

 * = Statistically significant.

¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) >5.66 (at p<0.05).

¨¨= Evaluated by T-test for independent samples. Significant at p<0.05.

 

D_n= Difference of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of D_n      β = slope of the curve       var(β) = variance of the slope

Solvent control = 1 (v/v) % Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

ND = No data (No cells were plated for colony growing due to excessive cytotoxicity observed during the treatment or in the expression period.)

 

Table 7.6.1/3: Mutagenicity Results of Assay 2 (3 hour treatment in the presence of S9-mix)

S9-mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn2/var(Dn)¨	Mutation frequency
+	3	2000 μg/mL	ND	ND	ND	ND	ND
		1750 μg/mL	ND	ND	ND	ND	ND
		1500 μg/mL	NE	NE	NE	NE	NE
		1250 μg/mL	NE	NE	NE	NE	NE
		1125 μg/mL	NE	NE	NE	NE	NE
		1000 μg/mL	599/768	101/768	68/768	4.041	111.9
		875 μg/mL	605/768	90/768	73/768	4.169	113.1
		750 μg/mL	609/768	90/768	69/768	3.302	107.2
		500 μg/mL	598/768	96/768	74/768	6.679	128.6*
		250 μg/mL	651/768	65/768	52/768	0.540	83.0
		125 μg/mL	668/768	60/768	40/768	0.028	71.7
		62.5 μg/mL	664/768	84/768	40/768	0.216	77.3
		31.25 μg/mL	625/768	94/768	49/768	2.095	98.5
		Solvent control	655/768	74/768	39/768	--	68.6
		Untreated control	648/768	68/768	52/768	--	79.2
		Positive control (CP: 4 μg/mL)	230/768	249/768	289/768	¨¨ 7.83E-11	956.0*

 In linear trend analysis β²/var (β) =8.61,significant.

 * = Statistically significant.

¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if D_n²/var(D_n) >5.66(at p<0.05).

¨¨= Evaluated by T-test for independent samples. Significant at p<0.05 compared to the untreated control.

D_n= Difference of log mutant frequency of dose “n” and that of the vehicle control

var(D_n) = variance of D_n      β = slope of the curve       var(β) = variance of the slope

Solvent control = 1 (v/v) % Dimethyl sulfoxide

CP = Cyclophosphamide

ND = No data (No cells were plated for colony growing due to excessive cytotoxicity observed during the treatment or in the expression period.)

NE = Not evaluated (Due to the high level of cytotoxicity.)

Table 7.6.1/4: Mutagenicity Results of Assay 2 (24 hour treatment in the absence of S9-mix)

S9-mix	Treatment period (hours)	Test item or control concentration	Number of empty wells/total number of wells	Number of large colonies/total number of wells	Number of small colonies/ total number of wells	Dn^2/var(Dn)¨	Mutation frequency
-	24	2000 μg/mL	ND	ND	ND	ND	ND
		1700 μg/mL	ND	ND	ND	ND	ND
		1500 μg/mL	ND	ND	ND	ND	ND
		1300 μg/mL	ND	ND	ND	ND	ND
		1100 μg/mL	ND	ND	ND	ND	ND
		900 μg/mL	664/768	71/768	33/768	0.006	93.8
		700 μg/mL	665/768	59/768	44/768	0.636	78.8
		500 μg/mL	663/768	69/768	36/768	1.721	69.7
		375 μg/mL	631/768	84/768	53/768	0.068	90.0
		250 μg/mL	672/768	66/768	30/768	1.335	71.9
		125 μg/mL	608/768	113/768	47/768	1.444	124.9
		62.5 μg/mL	631/768	97/768	40/768	0.068	90.0
		31.25 μg/mL	638/768	93/768	37/768	0.037	99.9
		Solvent control	620/768	96/768	52/768	--	95.6
		Untreated control	592/768	117/768	59/768	--	113.2
		Positive control (NQO: 0.1 μg/mL)	408/768	264/768	96/768	¨¨ 1.32E-07	343.5*

 

 

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the conditions of this Mouse Lymphoma Assay, no mutagenic effect of Anisole was observed either in the presence or in the absence of metabolic activation system.

Executive summary:

In a mammalian cell gene mutation assay (J. Hargitai, 2012) conducted according to the OECD 476 guideline and in compliance with GLP, mouse lymphoma L5178Y TK+/- 3.7.2 C cells cultured in vitro were exposed to Anisole at concentration ranging from 31.25 and 2000 µg/mL in the presence and absence of mammalian metabolic activation (S9 mix induced by Phenobarbitol/Beta-Naphthoflavone) in two independent experiments. A pre-test was performed in order to determine the concentration range for the mutagenicity experiments.

In experiment 1, following a 3-hour treatment with and without metabolic activation, the cytotoxicity of the test item was observed at 2000, 1750, 1500, 1250 and 1125 µg/mL concentrations. An evaluation was made using data of first surviving concentration of 1000 µg/mL and next seven lower concentrations (a total of eight samples).

In experiment 2, following a 3-hour treatment with metabolic activation, similarly to the first test, excessive cytotoxicity of the test item was observed at 2000, 1750, 1500, 1250 and 1125 µg/mL concentrations. An evaluation was made using data of first surviving concentration of 1000 µg/mL and next seven lower concentrations (a total of eight samples). In experiment 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed at 2000, 1700, 1500, 1300 and 1100 µg/mL concentrations, cells in these samples died during the treatment. Therefore, an evaluation was made using data of all examined concentrations from 900 µg/mL to 31.25 µg/mL (a total of eight samples).

In experiment 1 and 2, no insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments. There were no large changes in pH or osmolality after treatment.

 

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

The spontaneous mutation frequency of the negative (solvent) controls was in the recommended range in each test. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the solvent controls at the end of the expression period were within the acceptable range in all assays. The evaluated concentration ranges were considered to be adequate, as they covered the range from cytotoxicity to no or little cytotoxicity. The number of test concentrations met the acceptance criteria. Therefore, the overall study was considered to be valid.

 

In conclusion, under the test conditions of this study, Anisole was considered to be non-mutagenic to mammalian cells with and without metabolic activation.

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.

 

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to EU/OECD guidelines and GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

: due to technical reason, cells were maintained on 150 cm2 flask instead of 75 cm2 flasks before the experiments. This deviation was considered not to adversely affect the results or integrity of the study.

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2(R1), "Guidance on Genotoxicity Testing And Data Interpretation for Pharmaceuticals Intended For Human Use", 09 November 2011

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 (v/v) % Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 g/mL amphotericin-B) and 10 (v/v) % heat-inactivated fetal bovine serum (DMEM-10, culture medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 from the liver of rats treated with Phenobarbital and beta-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity assay:
0.76, 2.29, 6.86, 20.58, 61.73, 185.2, 555.6, 1666.7 and 5000 µg/mL (+/- S9-mix)
Chromosomal aberration test:
156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL (Assay 1 and Assay 2 with and without S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was soluble in Dimethyl sulfoxide (DMSO) at 500 mg/mL concentration. As this solvent is compatible to the test system, it was selected as solvent for the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

CP (with S9-mix), EMS (without S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no
- Exposure duration: 3 hours (with and without S9) / 20 hours (without S9)
- Expression time (cells in growth medium): 20 hours (with and without S9) / 28 hours (without S9)
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 23 hours (with and without S9) / 48 hours (without S9)

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): colchecine (2 - 2.5 hours before)


NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: Analysis of 200 metaphases / dose level was made, with 100 metaphases/culture

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (= % relative survival)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.

The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.

The historical control data were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.

Statistics:

For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

See Tables 7.6.1/1 and 7.6.1/2

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/ml (with S9-mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: measurement of pH was performed at the end of the treatment period in the preliminary test and in both main tests. No large changes in pH were detected.
- Effects of osmolality: measurement of osmolality was performed at the end of the treatment period in the preliminary test and in both main tests.
Changes in the osmolality were observed at the highest concentration (5000 µg/mL) in both experiments (+/- S9).
- Evaporation from medium: no data
- Precipitation: in Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 5000-625 µg/mL concentration range (experiment without metabolic activation) or 5000-1250 µg/mL concentration range (experiment with metabolic activation).
In Assay 2, similarly to the first experiment, insolubility was detected at the end of the treatment period in the final treatment medium in the 5000-1250 µg/mL concentration range in both experiments.

RANGE-FINDING/SCREENING STUDIES:
Two Concentration Selection Cytotoxicity Assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 3-hour treatment with metabolic activation or 20-hour treatment without metabolic activation, 28-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays, both in the absence and in the presence of a metabolic activation system. A total of nine test concentrations between 5000 and 0.76 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay.
No marked cytotoxicity was observed at all tested doses, therefore 5000 µg/ml was chosen as the highest concentration in the main test according
to the OECD guideline instructions.

COMPARISON WITH HISTORICAL CONTROL DATA:
Solvent and positive controls are in the range of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No marked cytotoxicty was observed in Assay 1 (a 3-hour treatment with metabolic activation and a 3-hour treatment without metabolic activation).
In Assay 2 (a 3-hour treatment with metabolic activation and a 20-hour treatment without metabolic activation), cytotoxicity was observed at 5000µg/mL in the presence of S9-mix (relative survival values was 29%).

Remarks on result:

other: strain/cell type: Chinese hamster lung fibroblasts (V79)

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/1:Summary table of Chromosome Aberration Assay 1

Concentration (μg/mL)	Time of Treatment / Sampling	Relative Survival^# (%)	Insolubility^##	Mean % aberrant cells^###
Anisole without metabolic activation (-S9)
Negative (solvent) control	3h / 20h	100	–	1.5
5000 µg/mL	3h / 20h	92	+	NE
2500 µg/mL	3h / 20h	61	+	NE
1250 µg/mL	3h / 20h	72	+	1.5
625 µg/mL	3h / 20h	69	+^a	1.5
312.5 µg/mL	3h / 20h	76	–	2.0
156.3 µg/mL	3h / 20h	76	–	NE
Positive control	3h / 20h	82	–	10.8***
Anisole with metabolic activation (+S9)
Negative (solvent) control	3h / 20h	100	–	1.5
5000 µg/mL	3h / 20h	71	+	NE
2500 µg/mL	3h / 20h	73	+	3.0
1250 µg/mL	3h / 20h	68	+^a	3.0
625 µg/mL	3h / 20h	62	–	1.0
312.5 µg/mL	3h / 20h	71	–	NE
156.3 µg/mL	3h / 20h	82	–	NE
Positive control	3h / 20h	54	–	75.0***

Negative (solvent) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 1 µL/mL

Positive control (+S9): Cyclophosphamide, 6 µg/mL

NE: not evaluated

#: compared to the negative (solvent) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: Minimal amount of precipitate was observed

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Table 7.6.1/2:Summary table ofChromosome Aberration Assay 2

Concentration (μg/mL)	Time of Treatment / Sampling	Relative Survival^# (%)	Insolubility^##	Mean % aberrant cells^###
Anisole without metabolic activation (-S9)
Negative (solvent) control	20h / 28h	100	–	1.3
5000 µg/mL	20h / 28h	69	+	2.0
2500 µg/mL	20h / 28h	77	+	3.5
1250 µg/mL	20h / 28h	95	+^a	1.5
625 µg/mL	20h / 28h	90	–	2.5
312.5 µg/mL	20h / 28h	82	–	NE
156.3 µg/mL	20h / 28h	83	–	NE
Positive control	20h / 28h	67	–	47.6***
Anisole with metabolic activation (+S9)
Negative (solvent) control	3h / 28h	100	–	5.5
5000 µg/mL	3h / 28h	29	+	3.0
2500 µg/mL	3h / 28h	62	+	4.0
1250 µg/mL	3h / 28h	85	+^a	5.0
625 µg/mL	3h / 28h	70	–	1.5
312.5 µg/mL	3h / 28h	83	–	NE
156.3 µg/mL	3h / 28h	70	–	NE
Positive control	3h / 28h	61	–	39.0***

Negative (solvent) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 0.4 µL/mL

Positive control (+S9): Cyclophosphamide, 6 µg/mL

NE: not evaluated

#: compared to the negative (solvent) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: Minimal amount of precipitate was observed

 ***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

Conclusions:

Interpretation of results:
negative (with and without metabolic activation)

Under the test conditions of this study, Anisole did not induce a significant level of chromosome aberrations with or without metabolic activation. Therefore, Anisole is considered not clastogenic in this test system.

Executive summary:

In an in vitro chromosome aberration test, performed according to OECD guideline N° 473 and in compliance with GLP, Chinese hamster V79 lung cells were treated with Anisole dissolved in DMSO in two independents experiments, both with and without a liver metabolizing system (S9-mix), obtained from rats previously treated with Phenobarbital and beta-naphthoflavone. The dose range was selected on the basis of the results of a preliminary toxicity test.

 

In Chromosome Aberration Assay 1, 3-hour treatment with metabolic activation and a 3-hour treatment without metabolic activation were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 5000, 2500, 1250, 625, 312.5 and 156.25 µg/mL.

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium in the 5000-625 µg/mL concentration range (experiment without metabolic activation) or 5000-1250 µg/mL concentration range (experiment with metabolic activation). There were no large changes in the pH, but higher than usual difference was observed in the osmolality at the highest concentration (5000 µg/mL) in both experiments. No marked cytotoxicity was observed in this assay. Therefore, concentrations of 1250, 625 and 312.5 µg/mL (a total of three) were chosen for evaluation in case of the experiment without metabolic activation; and 2500, 1250 and 625 µg/mL (a total of three) were chosen for evaluation in case of the experiment with metabolic activation. None of the evaluated treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in either experiment. Therefore, Assay 1 was considered to be negative.

 

In Chromosome Aberration Assay 2, 3-hour treatment with metabolic activation and a 20-hour treatment without metabolic activation were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 5000, 2500, 1250, 625, 312.5 and 156.25.

In Assay 2, similarly to the first experiment, insolubility was detected at the end of the treatment period in the final treatment medium in the 5000-1250 µg/mL concentration range in both experiments. No large changes in the pH were detected, but higher than usual difference was observed in the osmolality at the highest concentration (5000 µg/mL) in both experiments. Cytotoxicity was also observed at 5000 µg/mL concentration with metabolic activation (relative survival values was 29%). Therefore, concentrations of 5000, 2500, 1250 and 625 µg/mL (a total of four) were chosen for evaluation in both cases. None of the evaluated treatment concentrations caused a significant increase in the number of cells with structural chromosome aberrations in either experiment, thus Assay 2 confirmed the negative results of the first main test.

The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid metaphases were found in some cases in the negative (solvent) control, positive control or test item treated samples in the performed experiments. An endoreduplicated metaphase was detected for both positive control substances in Assay 1, no endoreduplicated metaphases were found in the samples of Assay 2.

 

The negative (solvent) control data were acceptable and in good correlation with the laboratory’s normal range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

 

In conclusion, Anisole did not induce a significant level of chromosome aberrations in the performed experiments with or without metabolic activation.Therefore, Anisole is considered not clastogenic in this test system.

 

This study is considered as acceptable and satisfies the requirements for the cytogenicity endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Reverse Mutation assay:

One reverse bacterial gene mutation test (Hargitai, 2012) was selected as a key study, (OECD 471, reliability 1). In this study, Anisole is not mutagenic in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strain WP2uvrA with and without metabolic activation, up to a limit concentration.These results are confirmed by a supporting study (Cinelli, 2000), (OECD 471 screening, reliability 2) providing negative results with or without metabolic activation in S. typhimurium strains TA98, TA100. Therefore, Anisole showed no mutagenic action in Bacteria.

In vitro Mammalian chromosome aberration test:

One fully reliable study is available (Hargitai, 2013) conducted according to OECD TG 473 and GLP (mammalian chromosome aberration, with and without liver microsomal activation, 3 and 20 hours treatment).

The dose-levels selected were 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL (Assay 1 and Assay 2 with and without S9-mix). The cytotoxicity was observed at 5000 µg/ml (with S9-mix).

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments, in at both harvest times and in absence or presence of S9 mix.

Therefore, Anisole is considered not to be clastogenic in this in vitro mammalian chromosome aberration assay.

 

In vitro Mammalian Cell Gene Mutation test:

One fully reliable study is available (Hargitai, 2012) conducted according to OECD TG 476 and GLP (mouse lymphoma assay, 3.25 – 1000 µg/mL, with and without liver microsomal activation, 3 and 24 h of treatment).

The test item did not induce mutations in the thymidine kinase locus assay using the mouse lymphoma cell line L5178Y up to the highest tested concentrations.

Therefore, Anisole is considered not to be mutagenic in this mouse lymphoma assay.

In vivo

No data available. Based on REGULATION (EC) No 1907/2006 as at July 2011 and the absence of positive results in the three above mentioned in vitro tests no additional testing for genetic toxicity in vivo is necessary.  

 

General: 

For each endpoint (bacterial gene mutation, mammalian gene mutation, & mammalian chromosome aberration) reliable, GLP compliant in vitro studies are available that all gave negative results.

Therefore it can be concluded that Anisole is neither clastogenic nor mutagenic.

Accordingly it can be concluded that Anisole is not genotoxic.

Justification for selection of genetic toxicity endpoint
No study was selected, since all in vitro studies were negative.

Short description of key information:
- Bacterial Reverse Mutation Assay: not mutagenic in bacteria (reliability 1, 2012)
- In vitro Mammalian Chromosome Aberration Test: not clastogenic (reliability 1, 2013)
- In vitro Mammalian Cell Gene Mutation Test: not mutagenic (reliability 1, 2012)

- Genetic toxicity in vivo: no data available

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Regarding the overall negative results from the in vitro genotoxicity studies, it is likely that Anisole doesn't present genotoxic activity potential; therefore no classification is required according to the CLP Regulation and to the Directive 67/548/EEC.