Isodecyl 3,5,5-trimethylhexanoate

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:

13 April 2017 - 1 September 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)

Version / remarks:

21 July 1997

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:

Isodecyl 3,5,5-trimethylhexanoate

EC Number:

261-674-1

EC Name:

Isodecyl 3,5,5-trimethylhexanoate

Cas Number:

59231-35-5

Molecular formula:

C19H38O2

IUPAC Name:

8-methylnonyl 3,5,5-trimethylhexanoate

Test material form:

liquid

Specific details on test material used for the study:

Isodecyl 3,5,5-trimethylhexanoate (CAS number 59231-35-5), batch number P7560, was a colourless liquid. It was received on 10 April 2017 and stored at 15-25°C, protected from light. Purity was stated as UVCB -100% (broad mixture of isomers) and the retest/expiry date was given as December 2018. The test article information provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor. Preliminary solubility data indicated that Isodecyl 3,5,5-trimethylhexanoate was soluble in dimethylformamide (DMF) up to at least 100 mg/mL. A maximum concentration of 5000 μg/plate was selected for Mutation Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997). A maximum concentration of 5000 μg/plate was also selected for Mutation Experiment 2. Test article stock solutions were prepared by formulating Isodecyl 3,5,5-trimethylhexanoate under subdued lighting in DMF with the aid of vortex mixing, to give the maximum required treatment concentration. The test article solutions were protected from light and used within approximately 4.5 hours of initial formulation.

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Mutation Experiment 1 (S-9 +-) ; 5, 16, 50, 160, 500, 1600, 5000 µg/plate
Mutation Experiment 2 (S-9 +-) ; 160, 300, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

DMF

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Test System
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, absence or presence of S-9 mix.

Mutation Experiments
Isodecyl 3,5,5-trimethylhexanoate was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony Enumeration Section 4.4). As the results of Mutation Experiment 1 were negative, treatments in the presence of S-9 in Mutation Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay. Volume additions for the Mutation Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (DMF) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Colony Enumeration
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as contamination affected the accuracy of the automated counter.

Analysis of Results
Treatment of Data
Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges (Sections 7.3 and 7.4). Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control plate counts were comparable with the historical control ranges. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate). However, adequate interpretation of biological relevance was of critical importance.

Acceptance Criteria
The assay was to be considered valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges as defined in Section 7.3
2. The positive control chemicals induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.

Evaluation criteria:

Evaluation Criteria
For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS
Toxicity, Solubility and Concentration Selection
Details of all treatment solution concentrations and final Isodecyl 3,5,5-trimethylhexanoate concentrations are provided in the Test Article Section 3.1.
Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Isodecyl 3,5,5-trimethylhexanoate at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, no evidence of toxicity, as would usually be manifest by a diminution of the background bacterial lawn and/or a marked reduction in revertant numbers, was observed.
Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160 - 5000 µg/plate, in order to examine more closely those concentrations of Isodecyl 3,5,5-trimethylhexanoate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.
The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Data Acceptability and Validity
The individual mutagenicity plate counts were averaged to give mean values, which are presented in Section 8. From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges (Section 7.3). The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle controls confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Mutation
Following Isodecyl 3,5,5-trimethylhexanoate treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Isodecyl 3,5,5-trimethylhexanoate mutagenic activity in this assay system.

Applicant's summary and conclusion

Conclusions:

It was concluded that Isodecyl 3,5,5-trimethylhexanoate did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

SUMMARY

Isodecyl 3,5,5-trimethylhexanoate was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

All Isodecyl 3,5,5-trimethylhexanoate treatments in this study were performed using formulations prepared in dimethylformamide (DMF).

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Isodecyl 3,5,5 -trimethylhexanoate at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, no evidence of toxicity was observed, as would normally be manifest as a thinning of the background bacterial lawn or a marked reduction in revertant numbers.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160 - 5000 µg/plate, in order to examine more closely those concentrations of Isodecyl 3,5,5-trimethylhexanoate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following Isodecyl 3,5,5-trimethylhexanoate treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Isodecyl 3,5,5 -trimethylhexanoate mutagenic activity in this assay system.

It was concluded that Isodecyl 3,5,5-trimethylhexanoate did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).