Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate

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:

Experimental start date 02 May 2019: Experimental completion date 21 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 002

Cas Number:

58394-64-2

Constituent 2

Reference substance name:

Reference substance 003

Cas Number:

103-23-1

Constituent 3

Reference substance name:

Reference substance 001

Cas Number:

2451-84-5

Test material form:

liquid

Remarks:

Clear colorless liquid

Details on test material:

Batch No.: CH180307A

Method

Target gene:

histidine or tryptophan locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fractions (CD Sprague-Dawley rats)

Test concentrations with justification for top dose:

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
The maximum concentration was 5000 µg/plate (the OECD TG 471 maximum recommended dose level).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

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 in-house. Dimethyl sulphoxide was therefore selected as the vehicle. The homogeneity and stability was previously confirmed for the test item in dimethyl sulphoxide formulations.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 Mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

80 µg/plate for TA1537,

Positive control substance:

9-aminoacridine

Remarks:

Without S9 Mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

0.2 µg/plate for TA98

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 Mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

5 µg/plate for TA98

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Details on test system and experimental conditions:

Test Item Preparation
The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in dry dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at room temperature. No correction for purity was required.
All formulations were used within four hours of preparation. Analysis was carried out in Experiment 1 to determine the concentration of the maximum test item formulation (50 mg/mL).

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle 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 overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously except that 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.
Incubation and Scoring
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. The plates were viewed microscopically for evidence of thinning (toxicity). Sporadic manual counts were performed due to spreading colonies, artifact interference and colonies on the edge of the plates which prevented an accurate automated count.

Test for Mutagenicity: 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).
Dose selection
Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.
Without Metabolic Activation
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 vehicle 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. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
With Metabolic Activation
The procedure was the same as described previously except that 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.
Incubation and Scoring
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. The plates were viewed microscopically for evidence of thinning (toxicity). Sporadic manual counts were performed due to light background contamination which prevented an accurate automated count.

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. 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 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that are statistically significant but are within the in-house historical vehicle/untreated control range are not reported in the tables section

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1 (plate incorporation)
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).
A test item precipitate (globular in appearance) was noted at and above 1500 g/plate in both the presence and absence of metabolic activation (S9-mix). This observation 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 metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). One statistically significant value was noted (TA1535 at 1.5 µg/plate in the absence of metabolic activation (S9-mix), however, this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

Experiment 2 (pre-incubation)
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).
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).
A test item precipitate (globular in appearance) was noted at and above 1500 µg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.
There were no 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).

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No 905-983-8) did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No 905983-8) was considered to be non-mutagenic.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item 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) was based on OECD TG 471 and was 1.5 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.  The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity of the test item following the change in test methodology.

Dose formulation analysis was carried out in Experiment 1 to determine the concentration of the test item  (maximum dose).  Homogeneity/stability analysis was also carried out on the test item. The test item formulations were shown to be stable and homogenous, the maximum concentration was found to be within the expected range.

Results

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 and without metabolic activation.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.  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 the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method). Similarly, 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).

A test item precipitate (globular in appearance) was noted at and above 1500 µg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation 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 metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).  One statistically significant value was noted (TA1535 at 1.5 µg/plate in the absence of metabolic activation (S9-mix); not identified as statistically significant on Table 2, as per the requirements of the Study Plan), however, this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

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).  

Conclusion

Reaction mass of benzyl 2-ethylhexyl adipate and bis(2-ethylhexyl) adipate and dibenzyl adipate (EC No 905-983-8) was considered to be non-mutagenic under the conditions of this test.