Benzoyl chloride, 3-methoxy-2-methyl-

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:

23 August 1996 - 20 September 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results. Only four bacterial strains were tested during the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 strains of S. typhimurium were used.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Remarks:

Deviation from GLP: Chemical analysis of dosing solutions was not performed, however, the initial stock solution of the test compound was prepared gravimetrically by an experienced investigator and subsequent dilutions were prepared volumetrically.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

427-440-1

EC Name:

-

Cas Number:

24487-91-0

Molecular formula:

C9H9ClO2

IUPAC Name:

2-Methyl-3-methoxy benzoyl chloride

Test material form:

not specified

Details on test material:

MMBC

Method

Target gene:

S. typhimurium: Histidine locus.

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The tester strains were subcultured in Oxoid Nutrient Broth #2 for approximately 10 hr at 37 (±1) °C. The fresh inoculum was used when bacterial growth was approximately 10^8 to 10^9 cells per mL.

Control plates were run to check for sterility, determine the background reversion rate and measure the response of each tester strain to a positive control compound.

Additional strain / cell type characteristics:

other: Strains were characterized for nutritional requirements, crystal violet sensitivity, and ampicillin resistance no more than 6 months prior to initiation of the study.

Metabolic activation:

with and without

Metabolic activation system:

S-9 activation, derived from the livers of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

0, 50, 200, 500, 2000 and 5000 µg/plate.

The initial stock solution of the test compound was prepared gravimetrically and subsequent dilutions were prepared volumetrically. Note: All concentrations were adjusted for active ingredient,

Vehicle / solvent:

The test substance was dissolved in acetone.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Details on test system and experimental conditions:

POSITIVE CONTROLS
In the presence of metabolic activation: 2 µg/plate 2-anthramine for all four strains.

In the absence of metabolic activation: 3 µg/plate 2-nitrofluorene for strain TA98; 2 µg/plate sodium azide (SA) for strains TA100 and TA1535; 100 µg/plate 9-aminoacridine for strain TA1537.

METABOLIC ACTIVATION
The S-9 used for metabolic activation was obtained from rats treated with Aroclor. The S-9 mix consisted of the following:

Nicotinamide-adenine dinucleotide phosphate (NADP) 4 mM
Glucose-6-phosphate (G-6-P) 5 mM
Magnesium chloride (MgCl2) 8 mM
Potassium chloride (KCl) 33 mM
Sodium phosphate buffer, pH 7.4 100 mM
Liver homogenate (S-9) from Aroclor 1254 treated rats 10 %

The phosphate buffer mix used in the non-activated portion of the assay consisted of the above mix with an equal volume of saline substituted for NADP and S-9.

MUTAGENESIS ASSAY
The tester strains were subcultured in Oxoid Nutrient Broth #2 for approximately 10 hours at 37 (±1) °C. The fresh inoculum was used when bacterial growth was approximately 10^8 to 10^9 cells per mL.

Control plates were run to check for sterility, determine the background reversion rate and measure the response of each tester strain to a positive control compound.

For the activated portion of the assay the following were added, in order, to sterile test tubes: 2 mL of top agar, 0.1 mL of the bacteria inoculum, 0. 1 mL of the appropriate concentration of test substance, and 0.5 mL of phosphate buffer mix (with S-9 and NADP). For the non-activated portion of the assay, the above procedure was followed, except that the 0.5 mL of phosphate buffer mix (without S-9 or NADP) was added to the tubes directly after addition of the top agar. Each test article concentration was tested in triplicate, in minimal plates (minimal-glucose agar medium). The controls were tested in six replicates in minimal plates. The contents of the tubes were mixed and poured onto petri dishes containing approximately 20 mL of the appropriate agar. Plates were allowed to set for several minutes then placed in covered plastic boxes and incubated at 37 (±1) °C for approximately 72 hours prior to colony counting.

Following the incubation period, sterility plates were checked for contamination. Following the sterility check, the number of colonies on each plate was determined. The mean and standard deviation for each concentration was calculated. Background growth was checked for each experimental point to observe any toxic response.

Evaluation criteria:

A mutagenicity assay is considered valid if the following conditions are met:

First, the spontaneous reversion rate, with and without metabolic activation, must be reasonably consistent with the expected range for the strain being used; i.e., 12-60 colonies per plate for TA98, 50-200 for TA100, 13-50 for TA1535, and 7-20 for TA1537. Second, the positive control materials must elicit a positive response. And third, strains must maintain their genotype, i.e., nutritional requirements, crystal violet sensitivity and ampicillin resistance.

A test substance is considered positive (mutagenic) if it elicits in independent assays a number of revertants per plate at least 2 times that observed in the solvent control (background). A response that does not meet this criterion but elicits a potential biologically significant response (e.g., a dose related increase in revertants per plate over 3 concentrations) is considered an equivocal response and requires further evaluation.

A test substance is considered negative (non-mutagenic) if the criteria for a positive assay were not met and the test substance was tested up to either 5,000 µg/plate, the limit of solubility, or the limit of toxicity. Toxicity is defined as the elimination of a uniform background lawn.

Statistics:

Statistical methods beyond the calculation of the mean and standard deviation are not considered necessary for the interpretation of this study.

Results and discussion

Test results

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The positive control article for TA100 without metabolic activation (sodium azide) did not elicit a positive response in the initial assay. The response was elevated, but under 2 fold. A confirmatory assay was conducted. The positive control article for TA100 without metabolic activation did elicit a positive response in the confirmatory assay. A mutagenic response was not detected in any of the Salmonella tester strains examined.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Definitive Assay

Dose level (µg/plate)	S-9	Mean Revertants/Plate and Standard deviation
		TA98	TA100	TA1535	TA1537
Controls
Acetone	+	26 6	276 14	18 2	14 3
Acetone	-	22† 3	246 33	19† 5	13 3
2-ANTH 2	+	826* 22	1152* 33	279* 13	209* 41
2-NF 3	-	492* 50	-	-	-
SA 2	-	-	466 12	594* 46	-
9-AA 100	-	-	-	-	163* 52
Test Substance
5000	+	26† 0	260 22	21 2	14 2
2000	+	28 1	266 23	20 1	13 5
500	+	25 4	274 24	18 4	11 1
200	+	26 6	262 16	19 4	15 5
50	+	30 3	283 47	15 5	12 2
5000	-	13 2	170 52	13 1	8 2
2000	-	17 5	250 62	13 1	9 2
500	-	19 2	229 35	17 4	16 5
200	-	17 6	235 48	17 2	11 5
50	-	23 6	229 32	19 4	11 2

*Positive Response (≥ 2 x solvent)

†Contaminated plate (excluded from calculations)

 

Table 2 Confirmatory Assay

Dose level (µg/plate)	S-9	Mean Revertants/Plate and Standard deviation
		TA98	TA100	TA1535	TA1537
Controls
Acetone	+	32 4	208 22	14 4	15 5
Acetone	-	29 5	200 12	22 3	13 3
2-ANTH 2	+	1194* 45	1335* 143	343* 18	257* 16
2-NF 3	-	698* 102	-	-	-
SA 2	-	-	504* 30	648* 26	-
9-AA 100	-	-	-	-	431* 98
Test Substance
5000	+	34 7	213 33	14 3	16 3
2000	+	35 5	221 9	14 6	15 4
500	+	31 4	229 8	16 0	15 4
200	+	32 6	235 7	14 3	11 3
50	+	30 2	236 15	14 4	13 2
5000	-	15 2	209 54	16 2	7 3
2000	-	20 4	263 36	24 3	12 1
500	-	28 7	229 50	19 5	12 2
200	-	25 4	200 29	20 2	14 5
50	-	26 10	202 32	16 3	14 6

*Positive Response (≥ 2 x solvent)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of the study, the test substance gave a negative (i.e. non mutagenic) response in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 in the presence and absence of metabolic activation.

Executive summary:

The potential of the test substance to cause gene mutation in bacterial strains was determined in accordance with standardised guidelines US EPA OPP 84 -2, OECD 4714 and EU Method B.13. Four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) were treated in the presence and absence of a rat liver derived metabolic activation system (S9 mix). The test substance was was evaluated at concentration ranging from 50 to 5000 µg/plate and the number of revertants was determined. The results were confirmed in an independent assay. During the study, the test substance did not induce an increase in revertants when compared to vehicle controls. This was true for all tester strains both with and without metabolic activation. Under the conditions of the study, the test substance was not mutagenic in the Salmonella gene mutation assay.