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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

 Bacterial mutation assays were carried out by Perstorp AB using NesterolTM, the trade name for isobutyric acid, monoester with 2,2,4-trimethylpentane-1,3-diol and in vitro mammalian studies by the Eastman Kodak Company using the trade name Texanol. All studies were performed according to GLP and guideline, and were therefore both considered to be key studies for this particular endpoint. The mutagenic/genotoxic potential of isobutyric acid, monoester with 2,2,4-trimethylpentane-1,3-diol has been characterized in a well conducted bacterial in vitro mutagenicity test and an in vivo mammalian bone marrow mouse micronucleus assay. In bacterial reverse mutation assays conducted by a method similar to OECD Guideline 471, there were no increases in mutation frequency in any strain of Salmonella typhimurium at concentrations up to 3000 or 5000 μg/plate, in the presence or absence of metabolic activation. The positive and negative controls in these studies induced the appropriate responses. The in vitro mammalian mutagenicity endpoint was done with an OECD 476 study in V79 cells and the HPRT locus. Cells were treated with test article up to the concentration resulting in precipitation, with and without metabolic activation. 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.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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.

Target gene:

HPRT

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

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.

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

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

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.

 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to GLPs and was similar to current OECD 471 guideline; purity not reported.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

OECD Guideline 471 also includes E.coli as one of the tester strains

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His (-)

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

No details provided in study report

Additional strain / cell type characteristics:

other: TA 1535 and TA 1537: uvr B and rfa mutations; TA 98 and TA 100: uvr B and rfa mutations, pkM 101 plasmid

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

No details provided in study report

Additional strain / cell type characteristics:

other: TA 1538: uvr B and rfa mutations

Metabolic activation:

with and without

Metabolic activation system:

Complete S9 mix contained: NADP (sodium salt) (4µM), D-glucose-6-phosphate (5µM), MgCl2 (8µM), KCl (33µM), Sodium Phosphate buffer (100µM), and S9 homogenate (80µL). S9 homogenate prepared from Sprague-Dawley adult male rat liver induced by Aroclor 1254.

Test concentrations with justification for top dose:

Initial Assay:
0.316, 1.0, 3.16, 10.0, 31.6, 100.0, 316.0, 1000, 3164, 10000 µg/plate

Mutagenicity Assay:
10.0, 100, 1000, 2000, 3000 µg/plate

Vehicle / solvent:

Dimethylsulphoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Sodium Azide used with tester strains TA 1535 and TA 100 at 0.5 µg/plate without metabolic activity.

Positive control substance:

9-aminoacridine

Remarks:

9-Aminoacridine used with tester strain TA 1537 at 75 µg/plate without metabolic activity.

Positive control substance:

other: Picrolonic acid

Remarks:

Picrolonic acid used with tester strain TA 1538 at 200 µg/plate without metabolic activity.

Positive control substance:

other: ICR-191

Remarks:

ICR-191 used with tester strain TA 98 at 5 µg/plate without metabolic activity.

Positive control substance:

other: 2-Aminoanthracene

Remarks:

2-Aminoanthracene used at 2.5 µg/plate (tester strains TA 1535 and TA 1537) and at 0.6 µg/plate (tester strains TA 98, TA 100, and TA 1538) with metabolic activity.

Details on test system and experimental conditions:

Every evening, all strains were cultured in nutrient broth for 16 hours at 37°C, resulting in 10^8 to 10^9 cells per mL. The following day all strains were examined for the appropriate genetic markers and for reversion to histidine prototrophy.

Positive controls:
Positive controls consisted of direct acting mutagens for nonactivation assays and of mutagens which required metabolic biotransformation for activation assays.

Negative/Vehicle controls:
Negative controls consisted of the solvent used in the top agar plus the other essential components.

Media:
The strains were cultured in Oxoid Medium 2 (Oxoid number CM67). The selective medium was Vogel-Bonner Medium E with 1.5% agar and 2.0% glucose (Litron Laboratories). The top agar consisted of 0.6% purified agar, 0.6% NaCl, 0.02 mM L-histidine, and 0.02 mM biotin.

Initial Toxicity Assay:
An initial screen was performed on the test substance to determine the dose concentrations for the mutagenicity assay. Ten successive half-log dilutions of the test substance in DMSO were prepared from a 100 mg/mL stock solution. The test substance (0.1 mL/plate) was tested in triplicate with TA 100 with and without metabolic activation at concentrations of 0.0003 - 10 mg/plate. Top agar consisted of 0.6% purified agar, 0.6% NaCl, 0.1 mM L-histidine, and 0.1 mM biotin. The plates once solidified were incubated at 37°C for 36-72 hours and the percent survival for each dose was determined.

Mutagenicity Assay:
The test substance (0.1 mL) was tested in triplicate with all tester strains with and without metabolic activation. The test substance with the appropriate tester strain was added to 2.0 mL of top agar, and the S9 activation mixture (or sodium phosphate buffer, where required). The mixture was poured over the Vogel-Bonner indicator plate. The plates once solidified were incubated at 37°C for 36-72 hours and the number of revertant colonies per plate was scored.

Evaluation criteria:

A test substance was considered positive to induce mutations when all of the following were met and there was a reproducible dose-response relationship which exceeded 10^-3 revertants per nanomole:
-Six replicates of the negative control were performed, number of revertants/plate were counted, and the mean (N) and standard deviation (Sn) calculated.
-The test substance was plated in triplicate and revertants per plate were counted. A mean (E) and standard deviation (Se) were calculated for each dose level. A formula derived from the Student’s t-test was used to determine if the mean for the test material was statistically significantly different from the negative control. To be considered positive, the mean of the test substance had to be greater than the mean of the negative control plus 3.5 times the standard deviation of the negative control [E > N + 3.5(Sn)].
-If the Negative controls were within +/- 1 standard deviation of the historical controls and one or more of the experimental results for the test substance were significant, a retest was performed.

Statistics:

The mean (N) and standard deviation (Sn) for the negative control were calculated and compared to historical control for each of the five strains.

The mean (E) and standard deviation (Se) were calculated from triplicate platings for each dose level. A formula derived from the Student’s t-test was used to determine statistical significance between the mean for the negative control (N) and the means for the test substance (E). If E> N +3.5 (Sn), then E was considered to be significantly different from the negative control N.

If significant positive results were found, the data were plotted as revertants/plate versus µg/plate and a linear regression analysis was performed.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Initial assay:
Texanol produced 100% toxicity to tester strain TA 100 at 3.16 mg/plate with and without metabolic activation. Toxicity was absent at the 1.0 mg/plate dose concentration.

Mutagenicity:
Texanol failed to produce an increased rate of reversion in any of the tester strains. Dose concentrations of 2.0 and 3.0 mg/plate produced varying degrees of toxicity to the background lawns, ranging from slight thinning to complete lethality. Positive and negative controls produced the proper responses.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It is concluded that, under the conditions of this test, 2, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate (Texanol) showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation, even at cytotoxic concentrations.

Based on an absence of genotoxic/mutagenic effects in this study, Texanol® Ester-Alcohol is not classified for “Germ Cell Mutagenicity” according to GHS.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 of S. typhimurium were exposed to 2, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate (Texanol) in DMSO at concentrations of 10.0, 100, 1000, 2000, and 3000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method. The positive controls induced the appropriate responses in the corresponding strains. There was no increased incidence in rate of reversion in response to the test substance for any of the tester strains used, either in the presence or absence of the rat liver S9 activation system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US Environmental Protection Agency, Method: HG-Gene Muta - S. typhimunum: The Salmonella typhimurium reverse mutation assay, 1984 guidelines.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His (-)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa, uvrB, and pKM101

Metabolic activation:

with and without

Metabolic activation system:

Complete S9 mix contained: NADP (sodium salt) (4mM), glucose-6-phosphate (5mM), MgCl2 (8mM), KCl (33mM), Sodium Phosphate buffer (100mM), and S9 homogenate (10% v/v). S9 homogenate prepared from Sprague-Dawley adult male rat liver induced by Aroclor 1254.

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate in the absence and presence of metabolic activation.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

2-Nitrofluorene (Aldrich, lot number 61896) was dissolved in DMSO at a dose concentration of 1 (TA98) µg/plate in the absence of metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

N-Ethy l-N-nitro-N-nitrosoguanidine (Sigma, lot number 67F-3700) was dissolved in DMSO at a dose concentration of 3 (TA100) and 5 (TA1535) µg/plate in the absence of metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

9-Aminoacridine (Sigma, lot number 96F-05641) was dissolved in DMSO at a dose concentration of 80 (TA1537) µg/plate in the absence of metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

2-Aminoanthracene (Aldrich, lot number 0013406) was dissolved in DMSO at dose concentrations of 2 (TA1535 and TA1537), 0.5 (TA98), and 1 (TA100) µg/plate in the presence of metabolic activation.

Details on test system and experimental conditions:

Preparation of S-9 fraction:
Approximately 7-8 week old Sprague-Dawley (Harlan Olac Ltd) rats were injected with a single intraperitoneal injection of Arochlor 1254 in Arachis oil at a dose level of 500 mg/kg/bw. On the fifth day after injection, rats were euthanized and the livers were removed at 0-4°C under aseptic procedures, placed in 0.15 M KCI (3ml KCl:1g liver), transferred to an Ultra-Turrax homogeniser and centrifuged at 9000 g for 10 minutes. The supernatant fraction (S-9 fraction) was stored at -80°C until required and the efficacy of each batch of S-9 fraction was tested with the carcinogens 7, 12-dimethylbenzanthracene and
2-aminoanthracene prior to use.

Cell culture growth:
An aliquot of frozen culture was added to 25 ml of nutrient broth (DAB 7, Merck) and incubated, with shaking, at 3°C for 10 hours. At least 2 X 10^9 cells per ml of each culture were checked photometrically and used on on the study.

Preliminary toxicity test:
Four concentrations of the test substance were assessed for toxicity using the tester strains with the highest concentration being 50 mg/ml of test substance in acetone providing a final concentration of 5000 µg/plate. Three 10-fold serial dilutions of the highest concentration were also tested. An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S-9 mix or 0.5 ml 0.1 M phosphate buffer (PH 7.4) were placed in glass bottles followed by an aliquot of 0.1 ml of the test solution then followed by 2 ml of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto prepared petri dishes containing 25 ml minimal agar with a single petri dish used for each dose level. Plates were also prepared without the addition of bacteria in order to assess
the sterility of the test substance, S-9 mix and phosphate buffer. Plates were incubated at 37°C for 3 days afterwards the plates were examined for the appearance of a background bacterial lawns and revertant colonies were counted using a Seescan Automatic Colony Counter.

Mutation test procedure:
Five concentrations of the test substance were assessed for toxicity using the tester strains with the highest concentration being 50 mg/ml of test substance in acetone providing a final concentration of 5000 µg/plate. Four half-log interval dilutions of the highest concentration were also tested. An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S-9 mix or 0.5 ml 0.1 M phosphate buffer (PH 7.4) were placed in glass bottles followed by an aliquot of 0.1 ml of the test solution then followed by 2 ml of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto prepared petri dishes containing 25 ml minimal agar with a single petri dish used for each dose level. A set of plates were also prepared containing only bacterial culture
and S-9 mix or phosphate buffer (0 µg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. Plates were incubated at 37°C for 3 days afterwards the plates were examined for the appearance of a background bacterial lawns and revertant colonies were counted using a Seescan Automatic Colony Counter. The assay was repeated in duplicate.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups was compared with the solvent control and the mutagenic activity of a test substance was assessed by the following criteria:
-If treatment produced an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it was considered to show evidence of mutagenic activity in this test system. No statistical analysis was performed.
-If treatment didn't reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it was considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.
-If the results obtained failed to satisfy the criteria for a positive or negative response the following was performed to resolve the issue of the substance's mutagenic activity:
a) Repeat tests using modifications of the experimental method that include (but are not restricted to), the use of a narrower dose range than
that already tested or to use different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed the substance was considered to show evidence of mutagenic activity. No statistical analysis was performed.
b) If no clear positive response can be obtained the data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers using the procedures described by Mahon et al. (1989).

Statistics:

If statistical analysis was performed then it followed the procedures of Mahon et al., (1989).

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
The revertant colony counts for Nesterol obtained in the preliminary toxicity test show Nesterol was toxic at the highest concentration towards three of the tester strains (TA1537, TA98 and T A 100) in the presence of S-9 mix and two (TA98 and TA100) in the absence of S-9 mix.

First mutation test:
Treatment with Nesterol in the first mutation test, toxicity was observed at the highest concentration towards TA98 and TA100 in the absence of S-9 mix and to TA1535, TA1537, TA98 and TA100 in the presence of S-9 mix.

Second mutation test:
Treatment with Nesterol in the second mutation test, toxicity was observed at the highest concentration towards TA1535 in the absence of S-9 mix and towards TA1537, TA98 and TA100 in the presence and absence of S-9 mix.

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with Nesterol at any dose level, in the presence or absence of S-9 mix, in either mutation test and the positive controls exhibited the proper responses.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It is concluded that, under the conditions of this test, 2, 2, 4-trimethyl-1, 3-pentanediol isobutyrate (Nesterol) showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation, even at cytotoxic concentrations.

Based on an absence of genotoxic/mutagenic effects in this study, Nesterol is not classified for “Germ Cell Mutagenicity” according to GHS.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535 and TA1537 of S. typhimurium were exposed to 2, 2, 4-trimethyl-1, 3-pentanediol isobutyrate (Nesterol) in acetone at concentrations of 50, 150, 500, 1500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method. The positive and negative controls induced the appropriate responses. There was no increased incidence in rate of reversion in response to the test substance for any of the tester strains used, either in the presence or absence of the rat liver S9 activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo mammalian bone marrow mouse micronucleus assay conducted by a method similar to that prescribed in OECD 474, there was no evidence of a significant increase in the number of micronuclei in bone marrow polychromatic erythrocytes of mice administered a single dose of 2000 mg/kg bw isobutyric acid, monoester with 2,2,4-trimethylpentane-1,3-diol , the highest dose tested. The vehicle and positive controls in both studies induced the appropriate responses.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: valid without restriction; GLP guideline study conducted by a method similar to OECD Guideline 474.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

-Strain/Source: Swiss CD-1, Crl:CD-1 (ICR)BR [Charles River Laboratories, Inc., St. Constant, Canada (LD50/3 phase) and Portage, MI (micronucleus phase)]
-Age (LD50/3 phase): Males, 76 days; Females, 68 days
-Age (micronucleus phase): Males, 66 days; Females, 58 days
-Body weight (LD50/3 phase): 26.7-31.8 g
-Body weight (micronucleus phase): 24.5-36.1 g
-Acclimatization: 5 days
-Housing: one or two per cage
-Temperature (°F) 72 ±3
-Humidity (%): 50 ± 20
-Photoperiod: 12 hours
-Feed: Agway Prolab RMH 3000 pellets ad libitum
-Water: Rochester, NY local municipality water ad libitum
-Randomization: randomly assigned to study groups based on individual body weights
-Identification: uniquely identified by ear tag

Route of administration:

oral: gavage

Vehicle:

Distilled water

Details on exposure:

LD50/3 phase:
A dose of 2000 mg/kg bw undiluted test substance was administered by gavage to three male and three female mice. Mice were observed for 24 hours.

Micronucleus phase:
Mice were administered the test substance via oral gavage at dose levels of 0, 200, 1000, and 2000 mg/kg bw. The negative control group was administered 2.11 mL/kg bw of distilled water while the positive control group received 80 mg/kg bw cyclophosphamide. Twenty-four hours after Texanol or negative control administration, one half of the animals were euthanized, femurs removed, and bone marrow smears prepared. Positive control animals were all euthanized at 24 hours. The remaining animals were euthanized 48 hours after test substance or negative control administration, femurs removed, and bone marrow smears prepared.

Duration of treatment / exposure:

Once

Frequency of treatment:

Once

Post exposure period:

24 (Texanol, negative control, and cyclophosphamide) and 48 hours (Texanol and negative control)

Remarks:

Doses / Concentrations:
2000 mg/kg bw Texanol
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw Texanol
Basis:
actual ingested

Remarks:

Doses / Concentrations:
200 mg/kg bw Texanol
Basis:
actual ingested

No. of animals per sex per dose:

5 animals/sex/dose:harvest time group

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control:
-Cyclophosphamide (Aldrich Chemical Co)
-Lot number: CW-05609LT
-Purity per supplier: >98%
-A sample analyzed by hplc before study initiation was found to be free of detectable impurities.

Tissues and cell types examined:

Slides were scored for micronuclei and the relative numbers of polychromatic (PCE) and normochromatic (NCE) cells. One thousand polychromatic cells per animal were routinely scored.

Details of tissue and slide preparation:

At harvest, the mice were euthanized with carbon dioxide and the adhering soft tissue and epiphyses of both femurs were removed. The marrow was flushed from the femurs, transferred to centrifuge tubes containing 2 mL fetal calf serum (1 tube/animal), centrifuged to create a pellet, supernatant removed, and the cells were resuspended. The suspension was spread on glass microscope slides and air dried.

The slides were then fixed in methanol, stained in Wright’s-Giemsa stain, rinsed in deionized water, allowed to air dry, and coverslipped using mounting medium. The slides were labelled in such a manner that the group was unknown to the examiner.

Evaluation criteria:

General:
The criteria for identification of micronuclei were those of Schmid (1976). The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE.

Interpretation:
A positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant increase in micronucleated PCEs for at least one dose level. A test substance that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative.

Bone marrow depression:
Bone marrow depression was defined as a statistically significant decrease in the percent PCE ([PCE/PCE + NCE] X 100) in treated animals compared to the corresponding negative controls.

Statistics:

The unit of scoring and statistical analysis was the micronucleated cell, not the micronucleus. The data were examined using plots, descriptive statistics, rank transformation, analysis of variance, Tukey’s HSD Test and Dunnett’s t-test. Statistical significance was detected using an alpha risk of ≤ 0.05.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The test substance produced transient signs of acute toxicity in the high-dose female mice.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Immediately after dosing, 2 of 10 high-dose females showed prostration, 4 of 10 exhibited lethargy, 1 of 10 was hypothermic, and another was ataxic. All animals were recovering by the 4-hr observation period, all appeared normal at 24 hours after dosing and remained healthy until the appropriate harvest times. All males, and the low- and mid-dose females appeared normal throughout the study.

The only statistically significant effect was a decrease in the number of micronucleated PCE’s when compared to controls in the 1000 mg/kg bw male mice at the 48 hour harvest time. Both sexes of mice treated with 80 mg/kg bw cyclophosphamide showed statistically significant increases in micronucleated PCEs and a statistically significant bone marrow depression at 24 hours when compared with the negative control.

Conclusions:

Interpretation of results (migrated information): negative
The test substance, 2, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate (Texanol) did not induce significant increases in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay in any dose group at any harvest time.

Based on an absence of genotoxic/mutagenic effects in this study, Texanol® Ester-Alcohol is not classified for “Germ Cell Mutagenicity” according to GHS.

Executive summary:

In an in vivo bone marrow micronucleus assay, groups of male and female CD-1 mice were administered a single dose of 2, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate (Texanol) by oral gavage at dose levels of 0, 200, 1000, and 2000 mg/kg bw. Animals dosed with the test substance were euthanized at 24 and 48 hours after dosing for extraction of the bone marrow. A distilled water control group was included at each harvest time and a positive control group, dosed with cyclophosphamide at 80 mg/kg bw, was included only at the 24-hr sampling time. At the highest dose tested, Texanol did not induce a statistically significant increase in micronuclei in bone marrow polychromatic erythrocytes or a significant level of bone marrow depression in either male or female mice, at either the 24-hr or 48-hr harvest time when compared with concurrent vehicle controls. The positive control, cyclophosphamide, induced highly significant increases in micronucleated polychromatic erythrocytes in both sexes of mice at 24 hours and significant bone marrow depression when compared with the controls. It is concluded that Texanol is negative in the in vivo mammalian bone marrow micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Although no in vitro or in vivo germ cell mutagenicity/genotoxicity studies were available for review, the total weight of evidence available indicates that isobutyric acid, monoester with 2,2,4 -trimethylpentane-1,3 -diol is not expected to induce heritable mutations in the germ cells of humans and is not classified for “Mutagenicity/genotoxicity” according to EU CLP.