3,5-dimethylbenzoyl chloride

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:

13 October 1992 - 8 March 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Principles of method if other than guideline:

Only 4 strains of Salmonella typhimurium were tested.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dimethylbenzoyl chloride

EC Number:

413-010-9

EC Name:

3,5-dimethylbenzoyl chloride

Cas Number:

6613-44-1

Molecular formula:

C9H9ClO

IUPAC Name:

3,5-dimethylbenzoyl chloride

Details on test material:

DMBC

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):

Strains were characterised 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 derived from the livers of rats induced with Aroclor 1254.

Test concentrations with justification for top dose:

Definitive Assay: 0, 50, 200, 500, 2000 and 5000 µg/plate.
Confirmatory Assay: 0, 160, 300, 500, 900 and 1600 µg/plate.

All concentrations were adjusted for active ingredient.

Vehicle / solvent:

Acetone

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

In the presence of metabolic activation for all 4 strains

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

In the absence of metabolic activation for TA98.

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In the absence of metabolic activation for TA100 and TA1535

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of metabolic activation for TA1537

Details on test system and experimental conditions:

Metabolic Activation
The S-9 used for metabolic activation was obtained from rats induced with Aroclor 1254. The S-9 mix consisted of the following:

Nicotinamide-adenine dinucleotide phosphate (NADP) 4 mM
Glucose-6-phosphate 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 induced rats 10 %

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

Mutagenesis Assay
The tester strains were sub-cultured in Oxoid Nutrient Broth #2 for approximately 10 h at 37 ± 1 degrees Celsius. 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, to determine the background reversion rate, and to 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 compound, 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 19 mL of the appropriate agar. Plates were allowed to set for several minutes then placed in covered plastic boxes and incubated at 37 ± 1 degrees Celsius for approximately 72 hours prior to colony counting. Results were confirmed in an independent assay.

Evaluation criteria:

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.
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 - 150 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 have maintained characteristics, i.e., nutritional requirements, crystal violet sensitivity and ampicillin resistance.
A test article 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 criteria 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 article is considered negative (non-mutagenic) if the criteria for a positive assay were not met and the test article was tested up to 5000 µg/plate or the limit of solubility or toxicity, whichever was lower. 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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the definitive assay, the test article was evaluated at doses of 50, 200, 500, 2000, and 5000 µg/plate (all concentrations adjusted for active ingredient) in the presence and absence of S-9 (see Table 1 for mean summary data).

The study was designed to evaluate the mutagenic potential of the test article up to the limits of solubility, toxicity, or 5000 µg/plate (whichever was lower). A precipitate was observed in all strains with metabolic activation at doses of 2000 µg/plate and greater, and in TA98 and TA1535 without metabolic activation at 2000 µg/plate. Precipitate and toxicity were observed in all strains without metabolic activation, at 5000 µg/plate and also at 2000 µg/plate in strain TA1537.
An independent confirmatory assay was performed at lower concentrations ranging from 160, 300, 500, 900 and 1600 µg/plate (see Table 2 for mean summary data). A precipitate was observed in strains TA98, TA1535 and TA1537 at 1600 µg/plate with metabolic activation, and in TA98 and TA1537 without metabolic activation. A mutagenic response was not detected in the tester strains in any of the experiments conducted.

At test article concentrations not exhibiting precipitate or toxicity, 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 Mean Summary Data

Data reported as: Mean (Standard Deviation)

Controls
	Mean Revertants/Plate
	S-9	TA98	TA100	TA1535	TA1537
Solvent Controls Acetone Acetone	+ -	24 (4) 40 (7)	125 (18) 126 (12)	14 (4) 17 (5)	8 (2) 13 (4)
Positive Controls 2-anthramine 2 µg/plate 2-nitrofluorine 3 µg/plate Sodium azide 2 µg/plate 9-aminoacridine 100 µg/plate	+ - - -	766 (113) * 377 (58)*	1314 (74)**   754 (55) *	134 (6) *   633 (53)*	86 (42)*     109 (66)*
Test Compound
Dose Level (µg/plate)	Mean Total Revertant Colonies/Plate
	S-9	TA98	TA100	TA1535	TA1537
5000 2000 500 200 50 5000 2000 500 200 50	+ + + + + - - - - -	-† -† 27 (6) 20 (3) 19 (2) -§ -† 25 (12) 36 (13) 37 (2)	-† 92 (30)‡ 121 (29) 101 (9) 124 (21) -§ 63 (12) 102 (24) 108 (10) 126 (12)	-† -† 18 (8) 19 (2) 15 (3) -§ -† 11 (2) 19 (1) 17 (3)	-† -† 2 (0) 8 (3) 9 (1) -§ -§ 7 (0) 14 (1) 17 (6)

*Positive response: ≥2 x solvent

**Misdosed

†Precipitate on plate

‡Precipitate on plate (1 out of 3)

§Toxicity and precipitate

**†‡§Observations excluded from calculations.

 

 

Table 2 Confirmatory Assay Mean Summary Data

Data reported as: Mean (Standard Deviation)

Controls
	Mean Revertants/Plate
	S-9	TA98	TA100	TA1535	TA1537
Solvent Controls Acetone Acetone	+ -	21 (3) 32 (6)	177 (6)# 184 (7)#	24 (4) 20 (4)	8 (2) 19 (1)
Positive Controls 2-anthramine 2 µg/plate 2-nitrofluorine 3 µg/plate Sodium azide 2 µg/plate 9-aminoacridine 100 µg/plate	+ - - -	961 (121)* 698 (154)*	1343 (116)*   669 (48)*	183 (30)   602 (64)*	121 938)*     143 (43)
Test Compound
Dose Level (µg/plate)	Mean Total Revertant Colonies/Plate
	S-9	TA98	TA100	TA1535	TA1537
1600 900 500 300 160 1600 900 500 300 160	+ + + + + - - - - -	-† 21 (7) 28 (7) 24 (7) 34 (8) -† 19 (1) 23 (10) 33 (9) 24 (4)	125 (43) 141 (13) 135 (18) 168 (11) 166 (20) 103 (20) 142 (8) 166 (1) 172 (10) 178 (20)	-† 11 (5) 15 (2) 20 (6) 16 (5) 13 (4) 17 (5) 14 (3) 17 (1) 23 (3)	-† 6 (2) 4 (2) 7 (3) 9 (2) -† 11 (3) 21 (5) 20 (4) 22 (6)

#Outside of historical database (3.0 Standard Deviations) (Due to a limited database: n = 6)

*Positive response: ≥2 x solvent

†Precipitate on plate. Observations excluded from calculations.

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, DMBC is not mutagenic in the Salmonella, gene mutation assay.

Executive summary:

DMBC was evaluated for mutagenic activity in the Salmonella typhimurium gene mutation assay (Ames test) in accordance with EPA 40 CFR Part 158.340 Guideline 84-2 and OECD Guideline 471.

 

Tester strains were TA98, TA100, TA1535, and TA1537 ± S-9. The test article was evaluated in a definitve assay at concentrations ranging from 50 to 5000 µg/plate (all concentrations adjusted for active ingredient) and the number of revertants was determined. An independent confirmatory assay was performed using doses ranging from 160 to 1600 µg/plate

The test article did not induce an increase in revertants when compared to solvent controls. This was true for all tester strains both with and without metabolic activation. Toxicity and/or precipitation of the test article were observed at concentrations of 2000 to 5000 µg/plate in the definitive assay. Precipitation of the test article was observed at a dose of 1600 µg/plate in strains TA98, TA1535 and TA1537 but not in TA100.

 

Under the conditions of this study, DMBC is not mutagenic in the Salmonella gene mutation assay.