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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: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test article, alkyl glycidyl ether, was received by Microbiological Associates, Inc. on 08/15/96 and was assigned the code number 96BK39. The test article was characterized by the Sponsor as a colorless mobile liquid that should be stored in a cool dry area in a closed container under nitrogen. An expiration date was not provided.
Upon receipt, the test article was described as a clear, colorless liquid and was stored at room temperature, under nitrogen, protected from exposure to light.

Method

Target gene:
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to base-pair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary tests: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, and 5000 ug/plate
Confirmatory assays:
TA98, TA 1535, TA 1537, (without activation) and TA 100, WP2 uvrA (with and without activation): 33, 100, 333, 1000, 5000 ug/plate
TA98, TA1535, (with activation): 10, 33, 100, 333, 1000, 3333 ug/plate
TA1537 (with activation): 3.3, 10, 33, 100, 333, 1000 ug/plate
Vehicle / solvent:
Ethanol was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in ethanol at approximately 500 mg/ml, the maximum concentration tested.
The vehicle used to deliver alkyl glycidyl ether to the test system was 100 % ethanol (EtOH), (CA5# 64-17-5), obtained from Pharmco Products, Inc.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoantracene, 2-nitrofluorene, sodium azide, 9-aminoacridine, methyl methanesulfonate
Details on test system and experimental conditions:
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli tester strain WP2 uvrA Salmonella tester strains were received on 11/10/92 directly from Dr. Bruce Ames, University of California, Berkeley. E. coli was received on 07/01/87 from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.
Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing -50 ml of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37±2·C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 109 cells per milliliter. The actual titers were
determined by viable count assays on nutrient agar plates.

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 was batch prepared on 04/25/96,07/03/96 and 08/07/96 and stored at ≤ 70·C until used. Each bulk preparation of S9 was assayed for its ability to metabolize 2-aminoantbracene and 7,12-dimethylbenz(a)antbracene to forms mutagenic to Salmonella typhimurium TAl00.
The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate,4 mM{3-nicotinamide-adenine dinucleotidephosphate,8 mMMg02 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 ml aliquot of each was plated on selective agar.
Evaluation criteria:
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article. Data sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
In the independent repeat assay, positive responses were observed with tester strain TA1535 in the presence (5.4-fold, maximum increase) and absence (4.1-fold, maximum increase) of S9 activation.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: Salmonella typhimurium histidine auxotrophs TA98, TA100, and TA1537
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:
In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/ml and a 50 µ1 plating aliquot. Generally, precipitate was observed at > 667 to >3333 µg per plate. Toxicity was generally observed at >1000 µg per plate in the presence of S9 activation only. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 µg per plate.
Mutagenicity Assay
The results of the mutagenicity assay were generated in Experiments Bl, B3 and B4. Generally, precipitate was observed at >1000 µg per plate. Toxicity was observed at >1000 µg per plate in the presence of S9 activation only. (Tables 26 and 27)
In Experiment B1, the mutagenicity assay, a positive response was observed with tester strain TA1535 (8.8-fold, maximum increase) in the presence S9 activation. Due to an unacceptable positive control value, tester strain TAl00 in the presence of S9 activation was not evaluated but was retested in Experiment B2.
Due to an unacceptable positive control value in Experiment B2, tester strain TAl00 in the presence of S9 activation was not evaluated but was retested in Experiment B4.
In Experiment B3, the independent repeat assay, positive responses were observed with tester strain TA1535 in the presence (5.4-fold, maximum increase) and absence (4.1-fold, maximum increase) of S9 activation. No other positive responses were observed with any of the remaining tester strain/activation combinations.
In Experiment B4, no positive response was observed with tester strain TAl00 in the presence of S9 activation.

In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/ml and a 50 µ1 plating aliquot. Generally, precipitate was observed at > 667 to >3333 µg per plate. Toxicity was generally observed at >1000 µg per plate in the presence of S9 activation only. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 µg per plate.
Mutagenicity Assay
The results of the mutagenicity assay were generated in Experiments Bl, B3 and B4. Generally, precipitate was observed at >1000 µg per plate. Toxicity was observed at >1000 µg per plate in the presence of S9 activation only. (Tables 26 and 27)
In Experiment B1, the mutagenicity assay, a positive response was observed with tester strain TA1535 (8.8-fold, maximum increase) in the presence S9 activation. Due to an unacceptable positive control value, tester strain TAl00 in the presence of S9 activation was not evaluated but was retested in Experiment B2.
Due to an unacceptable positive control value in Experiment B2, tester strain TAl00 in the presence of S9 activation was not evaluated but was retested in Experiment B4.
In Experiment B3, the independent repeat assay, positive responses were observed with tester strain TA1535 in the presence (5.4-fold, maximum increase) and absence (4.1-fold, maximum increase) of S9 activation. No other positive responses were observed with any of the remaining tester strain/activation combinations.
In Experiment B4, no positive response was observed with tester strain TAl00 in the presence of S9 activation.
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):
positive with metabolic activation tester strain TA1535
positive without metabolic activation tester strain TA1535
negative tester strains TA98, TA100, and TA1537

All criteria for a valid study were met as described in the· protocol. The results of the Bacterial Reverse Mutation Assay with an Independent Repeat Assay indicate that, under the conditions of this study, alkyl glycidyl ether did cause a positive response with tester strain TA1535 in the presence and absence of Aroclor-induced rat liver 59.
Executive summary:

The purpose of this study was to evaluate the mutagenic potential of the test article (or its metabolites) by measuring its ability to induce reverse mutations at selected loci of several strains ofSalmonella typhimuriumand one strain ofE. coliin the presence and absence of 59 activation.

The assay was performed in two phases, using the plate incorporation method. The first phase, the preliminary toxicity assay, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test article.

Ethanol was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in ethanol at approximately 500 mg/ml, the maximum concentration tested.

In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/ml and a 50 µl plating aliquot.

Generally, precipitate was observed at > 667 to >3333 µg per plate. Toxicity was generally observed at > 1000 µg per plate in the presence of 89 activation only. Based on the findings of the toxicity assay, the maximum dose plated in the mutagenicity assay was 5000 µg per plate.

In the mutagenicity assay, a positive response was observed with tester strain TA1535.

Generally, precipitate was observed at >1000 µg per plate. Toxicity was observed at >1000 µg per plate in the presence of 59 activation only.

Under the conditions of this study, test article alkyl glycidyl ether was concluded to be positive in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay.