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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: The study was conducted according to OECD Guideline 471 and GLPs.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
There was one deviation from the protocol. The deviation, however, was minor and did not affect the quality or integrity of the study.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test substance:
-Test substance (as cited in report): EC2000-0209, 2,2,4-trimethyl-1,3-pentanediol
-Lot number: 021500
-Date received: September 6, 2000
-Physical description: White crystalline chunks
-Storage conditions: Ambient temperature

Study dates:
-Initiation date: September 6, 2000
-Experimental start date: September 14, 2000
-Experimental termination date: October 23, 2000

Method

Target gene:
His (-), Trp (-)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The Salmonella typhimurium tester strains were received from Dr. Bruce Ames, Department of Biochemistry, University of California, Berkeley.
Additional strain / cell type characteristics:
other: TA 98 and TA 100: uvr B, rfa, pKM 101; TA 1535 and TA 1537: uvr B, rfa
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
The Escherichia coli tester strain was received from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).
Additional strain / cell type characteristics:
other: uvr A, pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate purchased from Molecular Toxicology, Inc., Lots 1081 and 1139 (44.2 and 40.8 mg protein/mL, respectively). The homogenate was prepared from male Sprague-Dawley rats injected (i.p.) with Aroclor 1254 (200 mg/mL in corn oil) at 500 mg/kg.
Test concentrations with justification for top dose:
Dose Rangefinding Assay (TA 100 and E. coli):
6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate

Mutagenicity Assay:
100, 333, 1000, 3330, and 5000 µg/plate

Vehicle / solvent:
Dimethyl sulphoxide (DMSO)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Benzo[a]pyrene (Sigma Chemical Company, purity ≥ 98%) was used with tester strain TA 98 at a concentration of 2.5 µg/plate with metabolic activation.
Positive control substance:
other: 2-Aminoanthracene
Remarks:
2-Aminoanthracene (Sigma Chemical Company, purity ≥ 98%) was used with tester strains TA 100, TA 1535, and TA 1537 at a concentration of 2.5 µg/plate and with tester strain WP2 uvr A (pKM 101) at a concentration of 5.0 µg/plate with metabolic activation.
Positive control substance:
2-nitrofluorene
Remarks:
2-Nitrofluorene (Sigma Chemical Company, purity ≥ 97%) was used with tester strain TA 98 at a concentration of 1.0 µg/plate without metabolic activation.
Positive control substance:
sodium azide
Remarks:
Sodium azide (Sigma Chemical Company, purity ≥ 98%) was used with tester strains TA 100 and TA 1535 at a concentration of 2.0 µg/plate without metabolic activation.
Positive control substance:
other: ICR-191
Remarks:
ICR-191 (Sigma Chemical Company, purity ≥ 90%) was used with tester strain TA 1537 at a concentration of 2.0 µg/plate without metabolic activation.
Positive control substance:
other: 4-Nitroquinoline-N-oxide
Remarks:
4-Nitroquinoline-N-oxide (Sigma Chemical Company, purity ≥ 99%) was used with tester strain WP2 uvrA (pKM 101) at a concentration of 2.0 µg/plate without metabolic activation.
Details on test system and experimental conditions:
Sterility Controls:
The most concentrated test substance dilution was checked for sterility by plating a 100 µL aliquot on selective agar. The S9 mix was checked for sterility by plating 0.5 mL on selective agar.

Vehicle controls:
Vehicle controls were plated for all tester strains in the presence and absence of S9 mix. The vehicle control was plated, using a 50 µL aliquot of DMSO, along with a 100 µL aliquot of the appropriate tester strain and a 500 µL aliquot of S9 mix (when necessary) on selective agar.

Preparation of S9 Metabolic Activation System:
The S9 homogenate was prepared from male Sprague-Dawley rats that had been injected (i.p.) with Aroclor 1254 at a dose level of 500 mg/kg bw. The S9 mix was prepared immediately prior to its use so that each mL contained the following components: H2O (0.70 mL), 1M NaH2PO4/Na2HPO4 (pH 7.4, 0.1 mL), 0.25 M glucose-6-phosphate (0.02 mL), 0.10M NADP (0.04 mL), 0.825M KCL/0.2 MgCl2 (0.04 mL), and S9 homogenate (0.10 mL ).

Dose Rangefinding study:
To determine cytotoxicity, a dose rangefinding study was performed on tester strains TA 100 and WP2 uvr A in the presence and absence of metabolic activation. Ten doses of TMPD were tested at one plate per dose up to a maximum of 5 mg/plate.

Mutagenicity Assay:
The assay was performed both in the presence and absence of S9 mix along with the appropriate vehicle and positive controls. Doses were chosen based on the rangefinding study. The results of the initial mutagenicity assay were confirmed in an independent experiment. All doses of the test article, the vehicle controls and the positive controls were plated in triplicate. The tester strains were exposed to the test substance via the plate incorporation methodology as described by Ames et al. (1975) and Maron and Ames (1983).

Plating Procedure:
When S9 mix was not required, 100 µL of tester strain and 50 µL of vehicle or test substance were added to 2.5 mL of molten selective top agar (45 ± 2 °C). When S9 mix was required, 500 µL of S9 mix, 100 µL of tester strain and 50 µL of vehicle or test substance were added to 2.0 mL of molten selective top agar. After the required components had been added, the mixture was vortexed, overlaid onto the surface of 25 mL minimal bottom agar and allowed to solidify. The plates were inverted and incubated for 52 ± 4 hours at 37± 2 °C. Positive control articles were plated using a 50 µL plating aliquot.

Plate Scoring and Counting:
Plates that were not evaluated immediately following the incubation period were held at 5 ± 3 °C. The numbers of revertant colonies per plate for the vehicle controls and all plates containing test article were counted manually. The numbers of revertant colonies per plate for the positive controls were counted by an automated colony counter.

Bacterial Background Lawn Evaluation:
The condition of the bacterial background lawn was evaluated both macroscopically and microscopically for indications of cytotoxicity and test substance precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate.

References:
Ames et al., 1975. Methods for detecting carcinogens and mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research, 31:347-364.

Maron DM and Ames B, 1983. Revised methods for the Salmonella Mutagenicity test. Mutation Research, 113:173-215.
Evaluation criteria:
Mutagenicity Assay: Before assay data were evaluated, the criteria for a valid assay must be met.
Assay Acceptance Criteria:
-All S. typhimurium tester strains with the rfa wall mutation must exhibit sensitivity to crystal violet.
-Cultures of tester strains TA 98, TA 100 and WP2uvr must exhibit resistance to ampicillin to demonstrate the presence of the pKM 101 plasmid.
-All vehicle control cultures must exhibit their characteristic number of spontaneous revertants. The acceptable values were: TA 98 (8-60), TA 100 (60-240), TA 1535 (4-45), TA 1537 (2-25), and WP2 uvr A (pKM 101) (80-350).
-To demonstrate that appropriate numbers of bacteria are plated, tester strain culture density must reach 0.5 X 10^9 bacteria/mL.
-Positive controls with and without metabolic activation must exhibit a 3-fold increase over the vehicle control for that strain.
-A minimum of 3 non-toxic doses were required to evaluate the assay data.

Assay Evaluation Criteria: Once criteria for a valid assay were met, responses observed in the assay were evaluated.
-Tester Strains TA 98, TA 100 and WP2 uvr A (pKM 101):
For a test substance to be considered positive, it must have produced at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains relative to the respective vehicle control and must be accompanied by a dose response to increasing concentrations of the test substance.
-Tester Strains TA 1535 and TA 1537:
For a test substance to be considered positive it must have produced at least a 3-fold increase in the mean revertants per plate of at least one of the strains relative to the respective vehicle control and must be accompanied by a dose response to increasing concentrations of the test substance.
Statistics:
For all replicate platings, the mean revertants per plates and the standard deviation were calculated.

Statistical analyses were not needed due to the absence of an increase in the number of revertant colonies at any dose level. Results for the positive control materials were in line with historical data on those substances.

Results and discussion

Test resultsopen allclose all
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:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Evaluation of Dosing Solutions:
The test material formed non-homogeneous suspensions in water down to concentrations of 29.9 mg/mL and a solution in water at 26.5 mg/mL but only with sonication and heating. Because the test material was more soluble in DMSO, forming a transparent colorless solution at up to 100 mg/mL, DMSO was selected as the vehicle

Dose Rangefinding study:
No cytotoxicity was observed with either tester strain in the presence or absence of metabolic activation as evidenced by the lack of a dose-related decrease in the number of revertants per plate and a normal background lawn. Therefore, the maximum concentration used in the rangefinding assay was selected as the highest dose level in the mutagenicity assay.

Mutagenicity Assay:
In the initial and the confirmatory mutagenicity assay, all data were acceptable and no positive increases were observed in the mean number of revertants per plate with any of the tester strains with or without metabolic activation. All criteria for a valid assay were met. It should be noted that because microbial contamination was observed on all the plates with tester strain TA 98 in the presence and absence of S9 mix in the confirmatory assay, TA 98 was retested. In the retest, all data were acceptable.

Deviation from protocol:
In the dose rangefinding assay, the vehicle control values for tester strains WP2 uvr A pKM 101 in the presence of S9 mix (388 revertants) and in the absence of S9 mix (362 revertants) were outside the acceptable range for this strain specified in the protocol (80-350 revertants). Since the rangefinding assay was performed only to assess the cytotoxicity of the test material to the test system, this deviation had no impact on the study. Since the values were only just outside the acceptable range, the data generated for the strain were acceptable in evaluating the toxicity of the test material to the test system.
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

At a maximum concentration of 5000 µg/plate, 2,2,4-trimethyl-1,3-pentanediol did not induce mutations in Salmonella typhimurium strains TA 98, TA 100, TA 1535 or TA 1537, or in E. coli strain WP2 uvrA pKM 101 when tested in the Salmonella/microsome bacterial mutagenicity assay (Ames assay) in the presence and absence of a metabolic activation system.

Based on the absence of genotoxic or mutagenic effects in this study with and without metabolic activation, 2,4-trimethyl-1,3-pentanediol is not classified for Germ Cell Mutagenicity according to the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) 1272/2008. 2,2,4-Trimethyl-1,3-pentanediol is not currently classified for Genetic Toxicity according to Directive 67/548/EEC.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and strain WP2 uvr A pKM 101 of E. coli were exposed to 2,2,4-trimethyl-1,3-pentanediol in DMSO at concentrations of 100, 333, 1000, 3330, and 5000 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 and there was no evidence of cytotoxicity or induction of mutant colonies over background. The results of the initial mutagenicity assay were confirmed in an independent second experiment.