Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-August-2021 to 27-August-2021

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

29 June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

31 March 2011

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1)

Version / remarks:

June 2012

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Molecular formula:

not applicable

IUPAC Name:

Reaction mass of benzoyl chloride, toluoyl chloride and 2-methyl resorcinol

Test material form:

solid: particulate/powder

Details on test material:

Appearance: beige powder

Specific details on test material used for the study:

Appearance: Beige powder
Storage conditions: Room temperature in the dark

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/plate.

The maximum concentration was 5000 µg/plate, the OECD TG 471 maximum recommended dose level.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Experiment 1 – Plate Incorporation Method
The maximum concentration was 5000 µg/plate. Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed for each tester strain, using the direct plate incorporation method.
-S9: A 0.1 mL aliquot of the appropriate concentration of test item, solvent or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlaid onto a Vogel-Bonner agar plate. Each concentration of the test item, appropriate positive and solvent controls and each bacterial strain, was assayed using triplicate plates. Untreated controls were also performed in triplicate on the same day as the mutation test.
+S9: The procedure was the same as described above except that untreated controls were not performed and, following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
The dose range used for Experiment 2 was 15, 50, 150, 500, 1500 and 5000 µg/plate.
-S9: A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, solvent or 0.1 mL of appropriate positive control were incubated at 37 ± 3°C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Each concentration of the test item, appropriate positive and solvent controls and each bacterial strain, was assayed using triplicate plates. Untreated controls were also performed in triplicate on the same day as the mutation test.
+S9: The procedure was the same as described above except that untreated controls were not performed and, following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3°C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

All of the plates were incubated at 37 ± 3°C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system.

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 andShelby, 1979); (2) A reproducible increase at one or more concentrations; (3) A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537.

A test item is considered non-mutagenic (negative) in the test system if the above criteria are
not met.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Precipitation observed at higher doses

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Precipitation observed at higher doses

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Precipitation observed at higher doses

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Precipitation observed at higher doses

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Precipitation observed at higher doses

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1:
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix. A precipitate of the test item was noted at and above 1500 µg/plate in both the presence and absence of S9-mix. This precipitate did not prevent the scoring of revertant colonies. There were no biologically relevant 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 S9-mix.

Experiment 2:
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix. A precipitate of the test item was noted at and above 500 µg/plate in both the presence and absence of S9-mix. This precipitate did not prevent the scoring of revertant colonies. There were no biologically relevant 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 S9-mix.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) BTMR did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix) and was thus considered to be non-mutagenic.

Executive summary:

Following the guideline OECD 471, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with BTMR using both the Ames plate incorporation and pre-incubation methods at up to eight 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 (plate incorporation) used 8 BTMR concentrations selected from the range 1.5 to 5000 µg/plate. As the overall result of Experiment 1 was Negative, the dose range of Experiment 2 was modified to six test item concentrations chosen from the range 15 to 5000 µg/plate.

The number of revertant counts for the solvent (dimethyl sulphoxide (DMSO)) control plates were 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the metabolic activation (S9-mix) were validated.

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix in Experiment 2.

A test item precipitate was noted at and above 1500 and 500 μg/plate in both the presence and absence of S9-mix in Experiments 1 and 2 respectively. The precipitate did not prevent the scoring of revertant colonies. There were no biologically relevant 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 S9-mix in both Experiment 1 and 2.

Under the conditions of this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) BTMR did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test BTMR was considered to be non-mutagenic.