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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Reliable without restriction; study was conducted according to GLPs and OECD 471, EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC Part B, Method B.14, and US Environmental Protection Agency, Method: HG-Gene Muta - S. typhimurium: The Salmonella typhimurium reverse mutation assay, 1984 guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
-Test material (as cited in report): Nesterol
-Chemical name (as cited in report):2,2,4-Trimethyl-1 ,3-pentanediol-isobutyrate
-Batch number: HRC 28.3.95/8
-Expiration date: September 28, 1995
-Purity: 99.5%
-Physical description: Colorless to light yellow liquid
-Storage: Room temperature

Method

Target gene:
His (-)
Species / strain
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
Controlsopen allclose all
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).

Results and discussion

Test results
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'.

Applicant's summary and conclusion

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.