2-Methoxy-4-methylphenyl methyl carbonate

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:

28-09-2011 to 17-11-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2011; signature: August 2011

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methoxy-4-methylphenyl methyl carbonate

EC Number:

700-673-7

Cas Number:

132638-45-0

Molecular formula:

C10H12O4

IUPAC Name:

2-methoxy-4-methylphenyl methyl carbonate

Test material form:

other: liquid

Method

Target gene:

histidine or tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/B-Naphthoflavone induced rat liver S9.

Test concentrations with justification for top dose:

Preliminary Toxicity Test (plate incorporation method): 0, 0.15, 0.50, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1 (plate incorporation method): 0, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 0, 50, 150, 500, 1500 and 5000 µg/plate
Dose levels were selected based on the results of the Preliminary Toxicity Test. The top dose was the guideline specified limit dose level selected due to absence of cytotoxicity and solubility of the test item in the solvent/vehicle.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed. Dimethyl sulphoxide was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With metabolic activation S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Experiment 2. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media. Subsequently, the procedure for incubation and duration was the same as in Experiment 1.

SELECTION AGENT (mutation assays): histidine-deficient agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.

Statistics:

Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).

Results and discussion

Test results

Species / strain:

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

Remarks:

See table 1 to table 4

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: cytotoxicity was not evident at any concentration

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1         Test Results: Range-Finding Test – Without Metabolic Activation

Test Substance Concentration (µg/plate)	Mean number of colonies per plate (SD)
	Base-pair substitution type			Frame-shift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
0	75 (14.5)	12 (1.5)	27 (4.2)	19 (7.0)	12 (3.5)
50	83 (4.0)	13 (4.2)	30 (1.0)	15 (1.0)	13 (1.0)
150	75 (1.0)	15 (1.5)	24 (2.1)	11 (1.7)	13 (1.5)
500	84 (3.2)	15 (3.2)	29 (0.6)	15 (2.6)	14 (0.6)
1500	72 (4.6)	15 (0.6)	24 (2.1)	12 (3.8)	11 (2.1)
5000	77 (0.6)	16 (2.5)	19 (6.8)	15 (3.8)	9 (2.1)
Positive Controls
Name	ENNG	ENNG	ENNG	4NQO	9AA
Concentration (µg/plate)	3	5	2	0.2	80
Mean number of colonies per plate (SD)	415 (33.1)	101 (15.5)	723 (9.8)	110 (11.3)	925 (13.5)

ENNG N-ethyl-N’-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine

Table 2         Test Results: Range-Finding Test – With Metabolic Activation

Test Substance Concentration (µg/plate)	Mean number of colonies per plate (SD)
	Base-pair substitution type			Frame-shift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
0	73 (10.2)	12 (1.2)	34 (2.3)	25 (1.2)	12 (5.2)
50	70 (5.6)	12 (3.1)	32 (0.6)	18 (3.1)	10 (1.5)
150	80 (5.9)	11 (3.5)	32 (2.5)	18 (0.0)	10 (0.0)
500	87 (20.8)	13 (1.5)	26 (1.0)	14 (0.6)	6 (0.6)
1500	77 (3.5)	12 (1.2)	24 (5.1)	11 (3.1)	15 (1.0)
5000	78 (10.7)	9 (3.5)	23 (3.2)	13 (2.3)	9 (2.1)
Positive Controls
Name	2AA	2AA	2AA	BP	2AA
Concentration (µg/plate)	1	2	10	5	2
Mean number of colonies per plate (SD)	720 (108.3)	336 (2.9)	269 (8.1)	295 (100.2)	256 (1.0)

BP Benzo(a)pyrene
2AA 2-Aminoanthracene

 

Table 3         Test Results: Main Test – Without Metabolic Activation

Test Substance Concentration (µg/plate)	Mean number of colonies per plate (SD)
	Base-pair substitution type			Frame-shift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
0	75 (13.5)	20 (8.5)	24 (4.5)	28 (2.5)	14 (4.0)
50	68 (7.0)	22 (8.7)	15 (2.0)	22 (4.5)	13 (4.6)
150	72 (3.2)	18 (3.2)	18 (6.9)	22 (1.5)	12 (3.0)
500	79 (11.6)	19 (5.0)	20 (2.1)	16 (2.0)	13 (2.1)
1500	65 (1.0)	16 (3.5)	14 (1.7)	23 (3.1)	7 (1.2)
5000	74 (16.1)	20 (6.1)	14 (3.0)	25 (9.0)	11 (4.5)
Positive Controls
Name	ENNG	ENNG	ENNG	4NQO	9AA
Concentration (µg/plate)	3	5	2	0.2	80
Mean number of colonies per plate (SD)	379 (33.8)	162 (12.5)	721 (40.5)	113 (0.6)	1393 (71.8)

ENNG N-ethyl-N’-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine

 

Table 4         Test Results: Main Test – With Metabolic Activation

Test Substance Concentration (µg/plate)	Mean number of colonies per plate (SD)
	Base-pair substitution type			Frame-shift type
	TA100	TA1535	WP2uvrA	TA98	TA1537
0	99 (10.2)	14 (3.8)	32 (5.6)	13 (3.2)	15 (3.1)
50	77 (12.3)	19 (4.5)	33 (3.0)	14 (3.1)	16 (2.5)
150	93 (10.3)	17 (8.5)	32 (5.1)	13 (1.7)	13 (2.9)
500	76 (6.6)	19 (2.1)	30 (0.6)	18 (7.1)	12 (3.6)
1500	88 (6.9)	16 (4.4)	25 (5.5)	15 (5.0)	15 (3.1)
5000	73 (14.8)	21 (1.7)	22 (10.4)	16 (2.9)	9 (1.0)
Positive Controls
Name	2AA	2AA	2AA	BP	2AA
Concentration (µg/plate)	1	2	10	5	2
Mean number of colonies per plate (SD)	707 (13.7)	389 (84.9)	198 (12.1)	248 (84.7)	247 (38.4)

BP Benzo(a)pyrene
2AA 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
Negative

Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using both the Ames plate incorporation and pre incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. Five test item dose levels were again selected in Experiment 2 and was 50 to 5000 µg/plate. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item caused no visible reduction in the growth of the bacterial lawn in all strains up to 5000 µg/plate. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9‑mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.