Reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate

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:

From 18 July to 3 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

Dimethyl adipate

EC Number:

211-020-6

EC Name:

Dimethyl adipate

Cas Number:

627-93-0

Molecular formula:

C8H14O4

IUPAC Name:

Dimethyl adipate

Constituent 2

Reference substance name:

Dimethyl glutarate

EC Number:

214-277-2

EC Name:

Dimethyl glutarate

Cas Number:

1119-40-0

Molecular formula:

C7H12O4

IUPAC Name:

dimethyl glutarate

Constituent 3

Reference substance name:

Dimethyl succinate

EC Number:

203-419-9

EC Name:

Dimethyl succinate

Cas Number:

106-65-0

Molecular formula:

C6H10O4

IUPAC Name:

dimethyl succinate

Test material form:

liquid

Remarks:

Colourless to brown liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:CY71353002
- Expiration date of the lot/batch: 01 June 2019
- Purity test date: 01 June 2017
- Analytical purity: 98.2%
- Commercial name of the registered substnace: Innroad Protect

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C)
- Stability under test conditions: Not assessed
- Solubility and stability of the test substance in the solvent/vehicle: DMSO was used as solvent to prepare the stock solution of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent and were used within 4 hours after preparation.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not checked

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Final dilution of a dissolved solid, stock liquid or gel: Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using DMSO

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base-pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA tryptophan (trp) reversion system measures trp- → trp+ reversions. The Escherichia coli WP2 uvrA detects mutagens that cause base-pair substitutions (AT to GC).

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial fraction (S9 fraction) from rat liver homogenate

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test. The maximum test concentration was 5000 μg test item/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO and Distilled water
- Justification for choice of solvent/vehicle: In the study two vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive control chemicals.Test item was insoluble at 100 mg/mL concentration using Distilled water. The test item was soluble at this concentration using DMSO and DMF. Due to the better biocompatibility, DMSO was selected as vehicle (solvent) for the study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water

True negative controls:

yes

Remarks:

i.e. untreated controls

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.
- Cell density at seeding (if applicable): not specified

DURATION
- Preincubation period: yes: 20 min at 37ºC in the Confirmatory Mutation Test (pre-incubation method)
- Exposure duration:48±1 hours at 37°C in the Initial Mutation Test (standard plate incorporation procedure) and the Confirmatory Mutation Test (pre-incubation method).
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

Evaluation criteria:

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
- at least five analysable concentrations are presented in all strains of the main tests;

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.

Statistics:

Not perfomed

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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

Cytotoxicity was reported only in the confirmatory mutation test at the concentration of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

Cytotoxicity was reported only in the confirmatory mutation test at the concentration of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

Cytotoxicity was reported only in the confirmatory mutation test at the concentration of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

Cytotoxicity was reported only in the confirmatory mutation test at the concentration of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative 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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

Cytotoxicity was reported only in the confirmatory mutation test at the concentration of 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

INITIAL AND CONFIRMATORY MUTATION TESTS:

In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used. The Initial Mutation

Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli

WP2 uvrA strain. The Initial Mutation Test and Confirmatory Mutation Test were performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.

Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate. In the Confirmatory Mutation Test, the test concentrations were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

No precipitate was observed in the main tests in all examined strains with and without metabolic activation.

In the Confirmatory Mutation Test, reduced/slightly reduced background lawn was observed in all examined strains on the plates at 5000 μg/plate concentration with and without metabolic activation.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent

controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 strain at 15.81 μg/plate

concentration without metabolic activation (the observed mutation factor value was: MF: 1.48). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

In the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA98 bacterial strain at 50 μg/plate

concentration with metabolic activation (the observed mutation factor value was: MF: 1.33). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no

background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used (Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA).
In conclusion, the registered substance reaction mass of diethyl adipate and diethyl glutarate and diethyl succinate has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item, Innroad Protect (commercial name of the registered substance), was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to the OECD No. 471 guideline.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and

TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the results of the Compatibility Test, the test item was dissolved in Dimethyl sulfoxide (DMSO) at a concentration of 100 mg/mL. Concentrations of

5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate. In the Confirmatory Mutation Test, the test item concentrations were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

No precipitate was observed in the main tests in all examined strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development) was observed in the Confirmatory Mutation Test in all examined strains with and without metabolic activation at 5000 μg/plate concentration. In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant

colonies did not show any biologically relevant increase compared to the solvent controls. There were no consistent dose-related trends and no indication of any

treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range and the reference mutagens showed the

expected increase in the number of revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable

concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be

valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes

or frameshifts in the genome of the strains used.

In conclusion, the registered substance Reation mass of diethyl adipate and diethyl glutarate and diethyl succinate has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.