Butanedioic acid, 2-hydroxy-, 1,4-bis[5,7,7-trimethyl-2-(1,3,3-trimethylbutyl)octyl] ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: other: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-03 to 2015-07-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The Bacterial Reverse Mutation Assay, also known as the Ames Assay, is used extensively to evaluate substances for mutagenic activity. The technique uses many different bacterial strains in order to compare the different changes in the genetic material. The result of the test detects the majority of genotoxic carcinogens and genetic changes; the types of mutations detected are frame shifts and base substitution.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

The method described in the above mentioned guidelines conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: bacterial reverse mutation assay (e.g. Ames test)

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] 2-hydroxybutanedioate

EC Number:

807-422-1

Cas Number:

66918-01-2

Molecular formula:

C40H78O5

IUPAC Name:

1,4-bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] 2-hydroxybutanedioate

Method

Target gene:

Histidine gene for auxotroph strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Tryptophan gene for auxotroph strain E. coli WP2 uvr A

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

Additional strain / cell type characteristics:

other: DNA exision repair-deficient

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial S9 fraction from livers of phenobarbital/β-naphthoflavone-induced rats

Test concentrations with justification for top dose:

- Preliminary Concentration Range Finding Test: concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
- Initial Mutation Test and Confirmatory Mutation Test: concentrations of 5000, 1581, 500, 158.1, 50, 15.81, 5 and and 1.581 µg/plate.

Vehicle / solvent:

Based the results of a solubility test, the test item was formulated in Acetone and the highest achievable concentration of the test item was 100 mg/mL in this vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone, DMSO and distilled water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD)

Details on test system and experimental conditions:

- Procedure for Exposure in the Initial Mutation Test
A standard plate incorporation procedure was performed as an Initial Mutation Test. Bacteria (cultured in Nutrient Broth No.2 ) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 μL
vehicle (solvent) or test item solution (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.

- Procedure for Exposure in the Confirmatory Mutation Test
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mutation Test no positive effect was observed.
Bacteria (cultured in Nutrient Broth No.2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, 50 μL of test item formulations (or its solvent, positive (reference) controls and their solvents), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. These tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.

Evaluation criteria:

The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
*Note: Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than two times higher than the reversion rate of the negative (solvent) control

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

According to the different guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Results and discussion

Test results

Test results 1

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Additional information on results:

In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used. In the main tests, each sample (including the controls) was tested in triplicate.

- In the Initial Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 1.581 μg/plate concentration with metabolic activation (the mutation factor value was 1.58). However, there was no dose response, the observed mutation factor value was below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range. Furthermore, higher numbers of revertant colonies compared to the Acetone control were observed for the untreated control (MF: 1.21) also in this strain with metabolic activation.
- In the Confirmatory Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA98 bacterial strain at 1581 μg/plate concentration with metabolic activation. The observed mutation factor value was 2.04. Although this value was slightly above the respective threshold value (MF=2) in this strain, there was no dose-dependent relationship (higher revertant counts compared to the negative (vehicle) control were observed at all other examined concentrations in this strain). The numbers of revertant colonies were within the historical control range in each case. Furthermore, the mutation factor value was lower than the threshold limit when compared to another controls of the same experimental day*. Thus, the observed values were considered as having no real biological relevance, just indicating higher than usual variability in this case.
*Note: The calculated mutation factor value is 1.37 when compared to the untreated control, and 1.73 when compared to the DMSO control.

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 test at some 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.

Precipitate (microdrops) was detected on the plates in the Initial Mutation Test in all examined bacterial strains at 5000 and 1581 μg/plate concentrations with and without metabolic activation; and in the Confirmatory Mutation Test in all examined bacterial strains at 5000 μg/plate concentration with and without metabolic activation and at 1581 μg/plate concentration with metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation No mutagenic activity, no inhibitory and cytotoxic effect
negative without metabolic activation No mutagenic activity, no inhibitory and cytotoxic effect

The test item Bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] malate was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. 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 metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test, an Initial Mutation Test and a Confirmatory Mutation Test. 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.
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 test item Bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] malate had no mutagenic activity in the examined bacterial strains under the test conditions of this study.

Executive summary:

The test item Bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] malate was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

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-naphthoflavone-induced rats.

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

Based the results of a solubility test, the test item was formulated in Acetone. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the preliminary experiment, the examined test item concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.

In the Initial Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In the Confirmatory Mutation Test, the observed revertant colony number was slightly above the respective biological threshold value in Salmonella typhimurium TA98 strain at 1581 μg/plate concentration with metabolic activation, but the calculated mutation factor value was below the threshold limit when compared to another controls of that experimental day. Additionally, there was no dose response and the numbers of revertant colonies were within the historical control range. Therefore, the observed colony counts were not considered to reflect a positive result.

Precipitate was detected on the plates in the Initial Mutation Test in all examined bacterial strains at 5000 and 1581 μg/plate concentrations with and without metabolic activation; and in the Confirmatory Mutation Test in all examined bacterial strains at 5000 μg/plate concentration with and without metabolic activation and at 1581 μg/plate concentration with metabolic activation.

No inhibitory, cytotoxic effect of the test item was observed in the main tests.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, 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 test, 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 test item Bis[2-(4,4-dimethylpentan-2-yl)-5,7,7-trimethyloctyl] malate had no mutagenic activity in the examined bacterial strains under the test conditions of this study.