Methyl anthranilate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from publication

Data source

Reference

Reference Type:

publication

Title:

Gene mutation toxicity study of the test chemical

Author:

Kristien Mortelmans et. al.

Year:

1986

Bibliographic source:

Environmental Mutagenesis, 1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Determination of Gene mutation toxicity study of the test chemical.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl o-aminobenzoate

Cas Number:

134-20-3

Molecular formula:

C8H9NO2

IUPAC Name:

Methyl o-aminobenzoate

Test material form:

liquid

Details on test material:

IUPAC name: 2-Aminobenzoic acid, methyl ester
Mol. formula: C8H9NO2
Molecular Weight: 151.1641 gm/mol
Smiles: c1(c(cccc1)N)C(OC)=O
InChI: 1S/C8H9NO2/c1-11-8(10)6-4-2-3-5-7(6)9/h2-5H,9H2,1H3

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

The S-9 mixes were prepared immediately prior to use and contained 10% RLI and 10% HLI respectively.
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9

- source of S9 : Male Sprague-Dawley rats and male Syrian hamsters

- method of preparation of S9 mix : Aroclor 1254 (200 mg/ml in corn oil) was administered ip at 500 mg/kg 5 days prior to decapitation (EGG, SRI) or cervical dislocation (CWR). The animals were deprived of food 12-24 hr immediately preceding death; otherwise food and water were provided ad libitum. The livers were removed aseptically, washed in ice-cold 0.15 M KCl, and minced and homogenized (3 ml of 0.15 M KC1 per gm of wet tissue) in a Potter-Elvehjem apparatus with a Teflon pestle. CWR initially used a Waring blender, but switched to a Potter-Elvehjem apparatus. The S-9 fraction was obtained by centrifugation of the liver homogenate for 10 min at 9,000 g at 4°C. The S-9 fraction was dispensed into freezing ampules and stored in a -70°C freezer, or in liquid nitrogen.

- concentration or volume of S9 mix and S9 in the final culture medium : 0.5 ml

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

Test concentrations with justification for top dose:

0.000, 33.000, 100.000, 333.000, 1000.000 and 1800.000 µg/plate

Vehicle / solvent:

Genetox: - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine for TA98, 2-aminoanthracene for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: At least five doses of the chemical were tested in triplicate. Experiments were repeated at least 1 wk following the initial trial

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER:
Preincubation method: The test chemical was assayed for mutagenicity in the preincubation assay. To each of 13 X 100-mm test tubes maintained at 37°C were added in the following order: 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37°C for 20 min, at which time 2.0 ml (CWR, SRI) of molten (45°C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 ml of minimal glucose bottom agar [Vogel and Bonner, 19561 in 15 X 100-mm plastic petri dishes. When the top agar had solidified, the plates were inverted and incubated at 37°C for 48 hr. Concurrent solvent and positive controls were tested with and without the metabolic activation systems. At least five dose levels of the chemicals were tested, with three plates per dose level. AU assays were repeated (as described above) no less than 1 wk after completion of the initial test.

Preliminary Dose-Setting Experiment:
The test chemical was initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his pin point
colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity. As a rule, at least one toxic dose was incorporated into the first mutagenicity test; the repeat test occasionally had the doses adjusted so that an apparent toxic dose was not reached.

Rationale for test conditions:

No Data Available

Evaluation criteria:

The plates were observed for a dose related increase in the number of revertants
The criteria used for data evaluation can be summarized as follows:
1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) non mutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.

Statistics:

No data available

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

Water

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data available

Remarks on result:

other: No mutagenic potenrial observed

Any other information on results incl. tables

Tables

1.

Dose	NA		TA100 10% HLI		10% RLI
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM
0.000	145	10.2	118	5.6	138	8.6
33.000	132	8.5	126	15.3	143	4.2
100.000	132	5.5	130	1.7	118	9.6
333.000	141	6.1	119	10.0	131	6.6
1000.000	130	5.3	110	5.5	121	9.6
1800.000	t	t	t		t
Positive control	1399	21.3	706	10.5	901	20.2

2.

Dose	NA		TA1535 10% HLI		10% RLI
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM
0.000	35	3.8	12	1.5	14	0.6
33.000	29	0.6	9	1.7	7	2.6
100.000	31	4.2	9	1.2	13	1.2
333.000	29	4.7	13	2.2	11	2.2
1000.000	13s	1.2	9	0.3	11	0.3
1800.000	t		t		t
Positive control	1316	14.5	64	8.7	108	5.5

3.

Dose	NA		TA1537 10% HLI		10% RLI
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM
0.000	7	0.9	5	0.9	5	1.2
33.000	5	1.7	8	1.2	7	1.3
100.000	7	0.6	7	1.5	9	1.5
333.000	8	1.2	8	3.0	8	1.2
1000.000	2	0.9	8	1.5	8	0.9
1800.000	t		6s	0.5	5s	1.0
Positive control	171	3.7	100	7.0	81	6.2

 

4.

Dose	NA		TA98 10% HLI		10% RLI
µg/plate	Mean	SEM	Mean	SEM	Mean	SEM
0.000	18	2.2	24	2.0	29	2.2
33.000	14	1.5	26	0.9	29	0.3
100.000	18	1.0	27	2.0	26	2.1
333.000	17	2.0	28	0.6	27	0.9
1000.000	13	1.2	24	2.1	24	1.5
1800.000	t		14s	0.9	t
Positive control	1267	44.6	949	94.4	670	26.3

 

t= toxic

s= slight toxic

Applicant's summary and conclusion

Conclusions:

The test chemical failed to induce mutation in the Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify for gene mutation in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1537, TA100 and TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 33, 100, 333, 1000 and 1800 µg/plate by the preincubation method. The liver S-9 was isolated from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters. Positive controls like sodium azide for TA1535 and TA 100, 4-nitro-o-phenylenediamine for TA98, and 9-aminoacridine for TA97 and TA1537; 2-aminoanthracene were used in the study. The test chemical failed to induce mutation in the Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify for gene mutation in vitro.