Isobutyric acid, monoester with 2,2,4-trimethylpentane-1,3-diol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Propanoic acid, 2-methyl-, monoester with 2,2,4-trimethyl-1,3-pentanediol

IUPAC Name:

Propanoic acid, 2-methyl-, monoester with 2,2,4-trimethyl-1,3-pentanediol

Constituent 2

Reference substance name:

Isobutyric acid, monoester with 2,2,4-trimethylpentane-1,3-diol

EC Number:

246-771-9

EC Name:

Isobutyric acid, monoester with 2,2,4-trimethylpentane-1,3-diol

Cas Number:

25265-77-4

Molecular formula:

C12H24O3

IUPAC Name:

3-hydroxy-2,2,4-trimethylpentyl 2-methylpropanoate

Constituent 3

Reference substance name:

(3-hydroxy-2,2,4-trimethylpentyl) 2-methylpropanoate

IUPAC Name:

(3-hydroxy-2,2,4-trimethylpentyl) 2-methylpropanoate

Constituent 4

Reference substance name:

Texanol Ester-Alcohol; TEXANOL; 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate

IUPAC Name:

Texanol Ester-Alcohol; TEXANOL; 2,2,4-trimethyl-1,3-pentanediol monoisobutyrate

Details on test material:

-Name of test material (as cited in study report): Isobutyric acid, 2,2,4-trimethyl-1,3-pentanediol monoester, mixed isomers

Specific details on test material used for the study:

Identification: Eastman Texanol (TM) Ester Alcohol
CAS Number: 25265-77-4
EC Number: 246-771-9
Batch Number: TXTXOL
Purity: 98.5%
Molecular Weight: 216.3
Expiry Date: 11 January 2023
Appearance: Clear colourless liquid
Storage Conditions: Room temperature, in the dark.

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Concentration µg/mL 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 ug/ml.
The maximum dose was set by data from the preliminary toxicity test where the results indicated that the maximum concentration should be the limited by test item precipitate, as recommended by the OECD 476 guideline

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Line
The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).

The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.

Cell Culture
The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 C with 5% CO2 in humidified air. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 µg/mL, 100 µM). Aminopterin (0.0178 µg/mL, 0.4 µM) and Thymidine (3.85 µg/mL, 16 µM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Microsomal Enzyme Fraction
The S9 Microsomal Enzyme Fraction was purchased from Moltox and Lot no 4370 with the expiry date of 24 November 2022 was used in the preliminary toxicology test and Lot no 4402 with the expiry date of 04 February 2023 for the main experiment of this study. The protein content was adjusted to approximately 20 mg/ml prior to use.
The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G­6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Main Experiment.

Evaluation criteria:

Assay Acceptance Criteria
The following criteria were used to determine a valid assay:

i) The background (spontaneous) mutant frequency of the vehicle controls is generally within the historical range. The background values for the with and without metabolic activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays.

ii) The concurrent positive controls should induce responses that are comparable with those generated in the historical positive control range and produce a toxicologically significant increase compared with the concurrent solvent control.

iii) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in a positive response.

iv) The criteria for selection of the maximum concentration have been met. The upper test item concentrations will be 10mM, 2 mg/mL or 2µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCBs) the upper concentration may need to be higher and the maximum concentration will be 5 mg/mL. Precipitating concentrations will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point. In the absence of precipitate and if toxicity occurs, the highest concentration should lower the relative survival (RS) to approximately 10 to 20 % of survival.

v) Adequate numbers of cells and concentrations are analysable. Mutant frequencies are normally derived from sets of ten dishes/flasks for the mutant colony count and three dishes for cloning efficiency counts. To allow for contamination losses / technical errors it is acceptable to score a minimum of eight mutant selection dishes and two cloning efficiency flasks.

vi) A minimum of four analysed concentrations is considered necessary in order to accept a single assay for evaluation of the test item.

Statistics:

Statistical analysis

When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances the mutant frequency was compared, where necessary, with the concurrent vehicle control value using the Chi squared Test on numbers of mutant colonies. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the mutant frequency was less than 0.05 and there was a dose-related increase.
The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Data tables are attached as a .PDF due to issues in pasting the table here.

Applicant's summary and conclusion

Executive summary:

Introduction

The purpose of this study is to assess the potential mutagenicity of a test item, supplied by the Sponsor, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Methods

Chinese hamster (V79) cells were treated with the test item at up to seven concentrations, in duplicate, together with solvent (dimethyl sulfoxide (DMSO)) and positive controls in the absence and presence of metabolic activation (S9).

The concentrations used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be the limited by test item precipitate, as recommended by the OECD 476 guideline. 

Results

The solvent (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.

The positive control treatments, both in the absence and presence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item did not induce any statistically significant increases in mutant frequency at any of the concentrations in either the absence or presence of metabolic activation. There was also no statistically significant concentration related increases when evaluated with a trend test, and all of the values observed were within the historical control range and 95% control limits for solvent controls. The results observed in both the absence and presence of metabolic activation were considered to fulfill the criteria for a clearly negative outcome.

Conclusion

The test item did not induce any toxicologically significant or dose-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. Eastman Texanol (TM) Ester Alcohol was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.