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Genetic toxicity in vitro

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

Endpoint:
in vitro gene mutation study in mammalian cells
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
The purpose of this study is to evaluate the mutagenic potential of the test article based on quantitation of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus of Chinese hamster ovary (CHO) cells.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO-K1-BH4. cells were obtained from Dr. Abraham Hsie, Oak Ridge National Laboratories, Oak Ridge, TN. CHO cells were cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT) then frozen. The freeze lot of cells was tested and found to be free of mycoplasma contamination. Cells used in the mutation assay were within four subpassages from frozen stock in order to assure karyotypic stability.
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:
9 concentrations of test article ranging from 0.5 to 5000 µg/mL in both the absence and presence of S9-activation.
Vehicle / solvent:
acetone (CAS 6764-1) obtained from Fisher Scientific
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: and ethylmethanesulphonate
Details on test system and experimental conditions:
Test System
CHO-K1-BH4. cells were obtained from Dr. Abraham Hsie, Oak Ridge National Laboratories, Oak Ridge, TN. CHO cells were cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT) then frozen. The freeze lot of cells was tested and found to be free of mycoplasma contamination. Cells used in the mutation assay were within four subpassages from frozen stock in order to assure karyotypic stability.

Metabolic Activation System
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 and stored at  -70QC until used. Each bulk preparation of S9 was assayed for sterility and its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz( a)anthracene to forms mutagenic to Salmonella typhimurium TA100.

Immediately prior to use, the S9 reaction mixture was prepared by mixing S9 and 10 mM calcium chloride (CaCl2) with a filter-sterilized cofactor pool to contain 100µL S9/mL cofactor pool, 4 mM nicotinamide adenine dinucleotide phosphate (NADP), 5 mM glucose-6-phosphate, 30 mM potassium chloride (KCl), 10 mM magnesium chloride (MgCI2), and 50 mM sodium phosphate buffer, pH 8.0. The S9 reaction mixture was stored on ice until used.
Evaluation criteria:
Evaluation of Test Results
The cytotoxic effects of each treatment condition were expressed relative to the solvent treated control (relative cloning efficiency). The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (usually 2xl0e6 cells: 10 plates at 2xl0e5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 10e6 clonable cells. For experimental conditions in which no mutant colonies were observed, mutant frequencies were expressed as less than the frequency obtained with one mutant colony. Mutant frequencies generated from doses giving 10% relative survival are presented in the data but were not considered as valid data points.

Because spontaneous mutant frequencies are very low for the CHO/HGPRT assay, calculation of mutagenic response in terms of fold increase in mutant frequency above the background rate does not provide a reliable indication of the significance of the observed response. The minimum mutant frequency for a response to be considered positive in this laboratory is conservatively set at >40 mutants per 10e6 clonable cells.
All conclusions were based on sound scientific judgement; however, the folloving criteria are presented as a guide to interpretation of the data:
• The test article was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least two consecutive doses showing mutant frequencies of > 40 mutants per 10e6 clonable cells.
• If a single point above 40 mutants per 10e6 clonable cells was observed at the highest dose, the assay was considered suspect.
• If no culture exhibited a mutant frequency of >40 mutants per 10 e6 clonable cells, the test article was considered negative.
Statistics:
The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (usually 2xl0e6 cells: 10 plates at 2xl0e5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection, and is expressed as TG-resistant mutants per 10e6 clonable cells.
Species / strain:
Chinese hamster Ovary (CHO)
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:
The test article was soluble in acetone at a concentration of 500 mg/mL, the maximum concentration tested.
Cloning efficiency relative to the solvent controls (RCE) was 18% at 5000 µg/mL without activation and 4% at 150 µg/mL with S9 activation. Based on the results of the toxicity test, the doses chosen for the initial mutagenesis assay ranged from 100 to 5000 µg/mL for the non-activated cultures and 25 to 150 µg/mL for the S9-activated cultures.

In the non-activated system of the initial mutagenesis assay, cultures treated with concentrations of 100, 250, 750, 2000 and 5000 µg/mL were cloned. In the S9-activated system, cultures treated with concentrations of 25, 50, 75, 100, 125 and 150 µg/mL were cloned. Test article precipitate was observed at a dose level of 5000 µg/mL in treatment medium. Relative cloning efficiency was 22% and 0% at the highest dose tested in the non-activated and S9-activated systems, respectively. None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 10e6 clonable cells. Based on these findings, the doses chosen for the independent repeat assay ranged from 50 to 5000 µg/mL for the non-activated cultures and 25 to 100 µg/mL for the S9-activated cultures.

In the non-activated system of the independent repeat assay, cultures treated with concentrations of 50, 100, 500, 2000 and 5000 µg/mL were cloned. In the S9-activated system, cultures treated with concentrations of 25, 50, 70, 80, 90 and 100 µg/mL were cloned. Test article precipitate was observed at dose levels of 2000 and 5000 µg/mL in treatment medium. Relative cloning efficiency was 16% and 51% at the highest dose tested in the non-activated and S9-activated systems, respectively. One S9-activated culture, treated with 100 µg/mL, exhibited a mutant frequency greater than 40 mutants per 10e6 clonable cells.

At the Sponsor's request the independent repeat assay was repeated over a range of 40 to 120 µg/mL with S9 activation. Cultures treated with concentrations of 40,60 and 80 µg/mL were cloned for mutant selection. No test article precipitate was observed at any dose levels in treatment medium. Relative cloning efficiency was 1% or less at cultures treated with 100, 110 and 120 µg/mL; these 3 cultures were not seeded for mutant selection. None of the selected cultures exhibited a mutant frequency greater than 40 mutants per 10e6 clonable cells.
Remarks on result:
other: strain/cell type: CHO-Kt-BH. cells
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, alkyl glycidyl ether was concluded to be negative with and without S9 activation.
Executive summary:

The purpose of this study is to evaluate the mutagenic potential of the test article based on quantitation of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus of Chinese hamster ovary (CHO) cells.

The test article, alkyl glycidyl ether, was tested in the CHO/HGPRT Mutation Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the dose range for the initial mutagenesis assay. The initial and independent repeat mutagenesis assay were used to evaluate the mutagenic potential of the test article.

 

Acetone was determined to be the solvent of choice based on solubility information from a previous study using the same test article (G96BK39.782001) and compatibility with the target cells. The test article was soluble in acetone at a concentration of 500 mg/mL, the maximum concentration tested.

 

In the preliminary toxicity assay, the maximum concentration of alkyl glycidyl ether tested was 5000 µg/mL. There was visible precipitate in the treatment medium at concentrations ≥500 µg/mL. Selection of dose levels for the mutagenesis assay was based on the cloning efficiency relative to the solvent control. Substantial toxicity, i.e. cloning efficiency£50% of the solvent control, was observed at concentrations of 1500 and 5000 µg/mL without activation and at concentrations ≥150 µg/mL with S9 activation. Based on these findings, the doses chosen for the initial mutagenesis assay ranged from 100 to 5000 µg/mL for the non-activated cultures and 25 to 150 µg/mL for the S9-activated cultures.

 

In the initial mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per 10e6 clonable cells, were observed. Visible precipitate was observed in treatment medium at a concentration of 5000 µg/mL. Concentrations of £2000 µg/mL were soluble in treatment medium. Toxicity, i.e., cloning efficiency£50% of the solvent control, was observed at doses of ≥100 µg/mL with and without S9 activation. Based on these findings, the doses chosen for the independent repeat assay ranged from 50 to 5000 µg/mL for the non-activated cultures and 25 to 150µg/mL for the S9-activated cultures.

 

In the independent repeat assay, one positive response was observed in the culture treated with 100 µg/mL with S9 activation. Visible precipitate was observed in treatment medium at concentrations of 5000 and 2000 µg/mL. Concentrations of £500 µg/mL were soluble in treatment medium. Toxicity was observed at doses of ≥500 µg/mL without activation and 90 and 100 µg/mL with S9 activation.

 

At the Sponsor's request the independent repeat assay was repeated over a range of 40 to 120 µg/mL with S9 activation. In the third assay with S9 activation, no positive responses were observed. No visible precipitate was observed in treatment medium at any concentration. Toxicity was observed at doses of ≥80 µg/mL with S9 activation.

 

In both mutagenesis assays without S9 activation, no cultures exhibited mutant frequencies of >40 mutants per 10e6 clonable cells. In the initial mutagenesis assay with S9 activation, the mutant frequency of the highest dose seeded for mutant selection was elevated, though it was not >40 mutants per 10e6clonablecells; in the independent repeat assay the high dose exhibited a mutant frequency> 40 mutants per 10e6clonablecells; in the third assay, no cultures exhibited mutant frequencies of >40 mutants per 10e6clonablecells. This indicates that the high mutant frequency exhibited in the independent repeat with S9 activation was probably an artifact. There was no dose response in any of the mutagenesis assays.

 

Therefore, the results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, alkyl glycidyl ether was concluded to be negative with and without S9 activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non-GLP study comparable to guideline study with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
Methods for detecting carcinogens and mutagens with the Salmonella/ Mammalian-Microsome mutagenicity Test. B.N. Ames. J. McCann and E. Yamasaki. Mutation Research 31: 347-364, 1975
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine dependence (auxotrophy) to histidine independence (prototrophy)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
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:
0.6 ug/plate, 1.85 ug/plate, 5.5 ug/plate, 16.7 ug/plate, 50 ug/plate and 150 ug/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: methylnitronitrosoguanidine for TA1535 and TA100, 9-aminoacridine for TA1537, 2-nitrofluorene for TA1538 and TA98, 2-aminoanthracene for all strain
Evaluation criteria:
Two fold or greater increase in reversion level over three doses.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
ambiguous with metabolic activation with TA100
ambiguous without metabolic activation with TA98
ambiguous with metabolic activation with TA1535

Ambiguous as the requirements for a significant dose-related effect over three concentrations with strain TA1535 and TA100 was not observed and a non-dose related two to three fold increase in the reversion of strain TA98 was observed.
Executive summary:

Salmonella Thyphimurium strain TA1535, TA1537, TA1538, TA98 and TA100 were tested for mutagenic activity in the salmonella Typhimurium Mammalian-Microsome mutagenic assay with and without metabolic activation system.

Results indicate that a limited response of strain TA1535 ranging from 3 to 4 fold at 50 and 150 ug/plate was produced by metabolism of the test agen to a weakly active form. About a two-fold increase in revertants of TA100 was also obtained at the same dose levels. Starin TA98 respoded in a similar manner, yielding approx. two to three-fold increases in revertant numbers at all test concentrations in the nonactivated test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non-GLP study comparable to guideline study with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Laboratory Procedure for Assessing Specific Locus Mutations at the TK Locus in Cultured L5178Y Mouse Lymphoma Cells. D. Clive and J.
Spector. Mutation Research 31 (1975) 17-29.
- A Mutational Assay System Using the Thymidine Kinase Locus in Mouse Lymphoma Cells. D. Clive, W. 6, Flamm, M. R. Machesko and N. J.
Bernheim. Mutation Research 16 (1972) 77-87.
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thimidine Kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Test concentrations with justification for top dose:
31.75 ug/mL, 62.5 ug/mL, 125 ug/mL, 250 ug/mL and 500 ug/mL.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulfonate that does not require activation to induce mutations, a promutagen (2-acetylaminofluorene) which depends on highly reactive S-9 for its mutagenic and toxi.c properties, and a promutagen (dimethylnitrósamine) that requires noninduce
Species / strain:
mouse lymphoma L5178Y cells
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
Conclusions:
Interpretation of results (migrated information):
negative

Under the experimetal conditions the substance R0069 did not showed any evidence of mutagenicity.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Treatment with the test item at dose levels up to 750 mg/kg/day did not cause biologically significant increases in MF at the cII gene in the liver or glandular stomach of Big Blue® male rats.

Although a statistically significant increase in MF (1.7-fold as compared to the vehicle control) was noted at 750 mg/kg/day for glandular stomach, this value was lower than the historical background mean (30.3 x 10-6 vs. 34.2 x 10-6 respectively).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non-GLP study comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
no
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
intraperitoneal
Vehicle:
mineral oil
Details on exposure:
The substance was injected intraperitoneally once a day during 5 days.
Duration of treatment / exposure:
5 days
Frequency of treatment:
once a day
Post exposure period:
1 day
Remarks:
Doses / Concentrations:
213.8 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
712.5 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2137.5 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females for the low, mid and high dose.
5 males and 5 females for the vehicle control and for the positive control.
Control animals:
yes, concurrent vehicle
Positive control(s):
Triethylene melamine was used as positive control substance. Single injection at day 5 at 0.04 mg/kg and a volume application of 5 mL/kg.
Tissues and cell types examined:
Bone marrow cells were collected from both femurs.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, R0069 administered intraperitoneally for five consecutive days at concentration as high as 712.5 mg/kg/day did not result in any consistent structural or numerical cytogenetic aberrations in rat bone marrow cells.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non-GLP study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Schmid, W. 1975. The micronucleus Test. Mutation Res. 31:9-15
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: B6D2F1
Sex:
female
Route of administration:
oral: gavage
Vehicle:
corn oil
Duration of treatment / exposure:
5 days
Frequency of treatment:
once a day
Post exposure period:
4 hours
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
ten females
Control animals:
yes, concurrent vehicle
Positive control(s):
Triethylmelanine
Tissues and cell types examined:
extraction of bone marrow
Statistics:
Dunnett's "t" test
Sex:
female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
The substance gave almost identical micronuclei values as the control and thus this chemical did not produce a mutagenic effect in terms of micronuclei at the dosage tested.
Endpoint:
in vivo mammalian somatic cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 May 2018 to 04 February 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)
Deviations:
yes
Remarks:
Refer to main study report
GLP compliance:
yes
Type of assay:
transgenic rodent mutagenicity assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: Huntsman Advanced Materials; Batch Number AAF1453400- Expiration date of the lot/batch: 26 November 2021- Purity test date: Not providedSTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature, protected from light- Stability under test conditions: Stable under the conditions indicated above for the period of use- Solubility and stability of the test substance in the solvent/vehicle: Soluble in corn oil at concentrations of 10, 25, 60 and 75 mg/mLOxirane formulated in corn oil, at concentrations of 1.02 and 76.9 mg/mL, was determined to be stable for at least 72 hours when stored at 2-8 degreesC and for at least 24 hours when stored at room temperature.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Test substance formulations were stirred with a magnetic stir bar until homogeneous in appearance prior to use
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
Rats have been used historically in safety evaluation and genotoxicity studies and are recommended by regulatory agencies. Because this study was conducted in accordance with regulatory guidelines, alternatives could not be considered.The F344 (wild-type) strain is the background strain of the Big Blue rats, and is appropriate and acceptable for use in the 5-day range finder study. For dose range-finding studies, it is not necessary to use transgenic rodents of the same strain.The Big Blue in vivo mutation assay is a Transgenic Rodent (TGR) Mutation assay described in OECD Test Guideline 488 (2013). TGR assays in general and the Big Blue assay in particular have been reviewed by the OECD and identified in OECD Test Guideline 488 as being appropriate to investigate in vivo mutagenicity in any tissue of interest. The TGR assays are also recommended to investigate a potential mutagenic mode of action in the etiology of rodent tumors.
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS - Source: In the 5-day range finder assay, wild-type Fischer 344 male rats (F344/NHsd) were received from Envigo, Frederick, MD. In the mutation assay, transgenic F344 Big Blue male rats were obtained from the BioReliance colony housed at Taconic Biosciences, Inc. (Germantown, NY). In addition, wild-type Fischer F344 male rats (F344/NHsd) were also received from Envigo, Frederick, MD for the TK cohort of the mutation assay. - Age at study initiation: Approximately 10 weeks old in the 5-day dose range finder assay; approximately 9 weeks old (mutation cohort) or approximately 8 weeks old (TK cohort) in the mutation assay - Weight at study initiation: 227.0 to 246.0 grams in the 5-day dose range finder assay; 188.1 to 232.5 grams (main study cohort) and 170.7 to 193.9 grams (TK cohort) in the mutation assay - Assigned to test groups randomly: [no/yes, under following basis: ] Yes, animals were randomized by body weight, using a computer-generated randomization program (Provantis Version 9.4.6.3), into dose groups. - Fasting period before study: None - Housing: Animals were housed in an environmentally-controlled room with continuous recording of room temperatures of 20.5 to 24.0 degreesC (approximately 69 to 75 degreesF) and a relative humidity of 30 to 70% with a 12-hour light/12-hour dark cycle (except when interrupted for study-related events). The animal rooms were supplied with at least 10 changes of HEPA-filtered air every hour. Animals for both the dose range-finder and mutation assays were multiple-housed during acclimation and following randomization, in polycarbonate cages. Sani-Chip hardwood bedding was used to absorb liquids. Cages and feeders were changed at least weekly. Animals were transferred to clean racks at least once every other week. Environmental enrichment was also provided in the form of Nylabones. - Diet (e.g. ad libitum): ad libitum, TEKLAD Global Diet #2018C (certified 18% protein rodent diet; Envigo, Madison, WI) - Water (e.g. ad libitum): ad libitum, via an automatic watering system (Napa Nectar gel packs were also provided, as needed) - Acclimation period: Animals were acclimated for 14 days in both the dose range-finder and mutation assays prior to the first dose administration. ENVIRONMENTAL CONDITIONS - Temperature (°C): 20.5 to 24.0 degreesC (approximately 69 to 75 degreesF) - Humidity (%): relative humidity of 30 to 70% - Air changes (per hr): The animal rooms were supplied with at least 10 changes of HEPA-filtered air every hour. - Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle (except when interrupted for study-related events) IN-LIFE DATES:5-day Dose Range FinderFrom: 15 May 2018 To: 02 June 2018Mutation AssayFrom: 12 June 2018 To: 23 July 2018 (TK cohort); 26 July 2018 (Mutation cohort)
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil - Justification for choice of solvent/vehicle: Corn oil was selected as the vehicle of choice based on the Sponsor's request and compatibility with the test system. - Concentration of test material in vehicle: 250, 500 and 750 mg/kg/day in the dose range-finder; 100, 250, 600 and 750 mg/kg/day in the mutation assay - Amount of vehicle (if gavage or dermal): 10 mL/kg - Lot/batch no. (if required): MKCC0462 and MKCD1021
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of Oxirane was weighed into a suitable size amber glass vial, calibrated with a PTFE stir bar (no correction facter was used). Approximately 70% of the vehicle was added to the vial while stirring. After mixing, the remaining amount of vehicle was added to the QS line, and the formulations were again stirred until homogeneous in appearance. Formulations were prepared daily for the dose range-finder and at least every three days for the mutation assay.
Duration of treatment / exposure:
In the dose range-finder assay, animals received the vehicle or test substance formulations once daily by oral gavage for 5 consecutive days. In the mutation assay, animals received the vehicle or test substance formulations once daily by oral gavage for up to 28 consecutive days.Tissues from positive control animals from a previous BioReliance study (AE53KK.171.BTL) were used. In the previous study, animals received ENU in buffer solution (pH 6.00) at 20 mg/kg/day, on Days 1, 2, 3, 12, 19 and 26, at a dose volume of 10 mL/kg.
Frequency of treatment:
Once daily
Post exposure period:
In the dose range-finder assay, animals were sacrificed on Day 5. In the mutation assay, animals in the TK cohort were bled for bioanalytical evaluation on Day 28 and sacrificed after completion of blood collection. Animals in the mutation cohort were sacrificed on Day 31.
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
Max dose indicated aboveAll doses in the mutation assay: 100.0, 250.0, 600.0, 750.0
No. of animals per sex per dose:
Dose range-finder: 3 animals/groupMutation assay: 6 animals/group (mutation cohort); 4 animals/group (TK cohort)
Control animals:
yes, concurrent vehicle
Positive control(s):
N-ethyl-N-nitrosourea - Justification for choice of positive control(s): ENU is a potent direct acting mutagen demonstrated to be mutagenic in the target tissues. The use of "packaging controls" is permitted by OECD Testing Guideline 488 as a way to reduce unnecessary animal usage. - Route of administration: Oral gavage - Doses / concentrations: Positive control animals from a previous BioReliance study (AE53KK.171.BTL) were used. In the previous study, animals received ENU in buffer solution (pH 6.00) at 20 mg/kg/day, on Days 1, 2, 3, 12, 19 and 26, at a dose volume of 10 mL/kg.
Tissues and cell types examined:
The liver and glandular stomach were collected from animals in the vehicle and test substance-treated groups for cII mutant analysis. These tissues were also analyzed from animals in the positive control study.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose levels for the 5-day dose range-finder were selected in consultation with the Sponsor, based on toxicity data available to the Sponsor, and a limit dose of 1000 mg/kg/day. Dose levels were also selected with consideration to dose selection criteria discussed in ICH S2(R1) for repeat dose studies longer than 14 days and OECD Testing Guideline 407 for 28-day repeat dose toxicity studies.Dose levels for the mutation assay were selected based on the results of the 5-day dose range-finder. TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): In the dose range-finder assay, animals received the vehicle or test substance formulations once daily by oral gavage for 5 consecutive days and were sacrificed on Day 5. No necropsy was performed following scheduled sacrifice.In the mutation assay, animals received the vehicle or test substance formulations once daily by oral gavage for up to 28 consecutive days. Animals in the TK cohort were bled for bioanalytical evaluation on Day 28 and sacrificed after completion of blood collection. Animals in the mutation cohort were sacrificed on Day 31 and tissues were collected for cII mutant analysis. METHOD OF ANALYSIS: Liver and glandular stomach tissue samples were processed for DNA isolation from frozen tissues of the first 5 animals in the control group (Group 1) and three selected test substance-treated groups (Groups 2, 3 and 5 only; these groups were selected by the Study Director in consultation with the Sponsor). Tissues from the remaining animals/groups were retained frozen as reserve samples, if needed. Positive control tissues were extracted and DNA was used for packaging. Tissues were extracted following BioReliance SOPs, based on methods described for Agilent product RecoverEase for somatic tissues. Isolated DNA samples were stored at 2-8 degreesC.Isolated DNA was processed using Packaging Reaction Mix (PRM), purchased from New York University (New York, NY). This product is similar to Transpack manufactured by Agilent, Santa Clara, CA. PRM is used to isolate recoverable lambda shuttle DNA vectors from the genomic DNA and to package lambda shuttle vector DNA, using phage proteins and cofactors to create infectious lambda phage particles. Methods followed BioReliance SOPs, based on Agilent instruction manual titled "Lambda Select-cII Mutation Detection System for Big Blue Rodents" and the Agilent instruction manual titled "Transpack Packaging Extract for Lambda Transgenic Shuttle Vector Recovery".Packaged phage were incubated overnight at 37 +/- 1.0 degreesC, and then scored for plaque formation and titer determination; cII mutant selection plates were incubated for two days (between 40 to 48 hours) at 24 +/- 0.5 degreesC, and then scored for mutant plaques. At least 125,000 phage were evaluated from at least 2 packagings for each dose and tissue. Titer and mutant frequency were calculated per BIoReliance SOP.
Evaluation criteria:
The test substance was considered to have produced a positive response if it induces a statistically significant increase in the frequency of cII mutants in any dose level, outside the 95% control limits of the historical background mutant frequency range.The test substance was considered to have produced a negative response if no significant increase in cII mutant frequency was observed.Equivocal responses (if noted) were evaluated by the Study Director on a case-by-case basis, considering both statistical significance and biological relevance.Other criteria may have also been used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological relevance and plausibility, a qualitative dose-related increase). Any such considerations, if used, were clearly reported and described by the Study Director.
Statistics:
The incidence of all effects was analyzed separately by dose level. Dunnett's test was conducted on body weight, body weight changes and organ weight data. All statistics compared treated groups versus the concurrent control (Group 1) and were based on a significance value of p< 0.05.The individual animal is considered the experimental unit. Mutant frequency was calculated (number of mutant phage/number of total phage screened) for each tissue analyzed from each animal. Since this ratio is extremely small and may not be normally distributed, a log10 transformation of the MF data was performed.The statistical analysis of MF was conducted in two parts. Initially, the positive control group was compared to the vehicle control group. In the second part of the analysis, all treated groups (except the positive control) were compared to the vehicle control. In both instances, log10 transformed MF data was evaluated using 1-Way Analysis of Variance (ANOVA). The suitability of using the parametric ANOVA was confirmed by testing parameters of the log10 transformed MF data for normality and equal variance. If the data appeared normally distributed and exhibited equal variance, the parametric ANOVA analysis was used; if either test failed, a non-parametric method was used.Statistical analysis was performed using Minitab® 16.1.0.Further details are outlined in Appendix F of the report.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Dose-range finder: ruffled fur sporadically for one or two animals/group; Mutation asay (sporadically seen): hunched posture, piloerection for all animals (cage-side); hunched posture, ruffled fur for all animals (hands-on); some decreased motor activity.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY - Dose range: 250, 500, 750 mg/kg/day - Clinical signs of toxicity in test animals: During cage-side observations, ruffled fur was recorded for one or two animals/group, starting at 250 mg/kg/day, on Day 5 only. No other clinical signs were noted during cage-side or hands-on observations. RESULTS OF DEFINITIVE STUDY - Statistical evaluation: No biologically significant elevated MF was observed at the cII gene in the liver or glandular stomach of Big Blue male rats. The only statistically significant increase in MF (1.7-fold as compared to the vehicle control) was noted at 750 mg/kg/day for glandular stomach; however, the mean MF value at this dose level was slightly lower than the historical background value. Since individual MF values for all animals treated at 750 mg/kg/day were within the 95% CL of the historical background data, this result was not considered biologically significant.
Conclusions:
Treatment with the test item at dose levels up to 750 mg/kg/day did not cause biologically significant increases in MF at the cII gene in the liver or glandular stomach of Big Blue® male rats.
Although a statistically significant increase in MF (1.7-fold as compared to the vehicle control) was noted at 750 mg/kg/day for glandular stomach, this value was lower than the historical background mean (30.3 x 10-6 vs. 34.2 x 10-6 respectively).
Executive summary:

This study investigated the effect of Oxirane on mutant frequency at the cII gene in liver and glandular stomach from male transgenic Fischer 344 Big Blue® rats; the study also included a 5day dose range-finding study. The Big Blue® Assay is a Transgenic Rodent (TGR) Mutation assay described in OECD Test Guideline 488 (OECD, 2013).

The 5-day range-finding study consisted of 12 wild-type Fischer 344 (F344) male rats (3/group). Group 1 animals received the vehicle (corn oil) and Groups 2 through 4 received Oxirane in the vehicle at doses of 250, 500, and 750 mg/kg/day, respectively, once daily by oral gavage for 5 consecutive days, at a dose volume of 10 mL/kg. Animals were sacrificed on Day 5; no necropsy was performed following scheduled sacrifice.

The mutation assay cohort consisted of a main cohort of 30 transgenic Fischer 344 Big Blue® male rats (6/group) and a TK cohort of 20 wild-type F344 male rats (4/group). Vehicle control animals (Group 1) were treated with corn oil. The test substance treated animals (Groups 2, 3, 4 and 5) were treated with Oxirane in the vehicle at doses of 100, 250, 600 and 750 mg/kg/day, respectively. Animals in Groups 1-5 were dosed once daily via oral gavage for up to 28 consecutive days. As part of a previous BioReliance study (AE53KK.171.BTL), six positive control animals for the Big Blue® assay (designated as Group 6) were treated with N-ethyl-N-nitrosourea (ENU) in buffer solution (pH 6.00) at 20 mg/kg/day, on Days 1, 2, 3, 12, 19, and 26. All doses were administered at a dose volume of 10 mL/kg.

Animals in the TK cohort were bled from the retro-orbital sinus (under 70% CO2/30% O2 anesthesia) on Day 28, for bioanalytical evaluation. After completion of blood collection, animals in this cohort were sacrificed by CO2 overdose and discarded without necropsy. Animals from the mutation assay cohort were sacrificed by CO2 overdose on Day 31. A partial necropsy was performed for animals in Groups 1-5; the liver and glandular stomach were collected for cII mutant analysis; the testes and duodenum were also collected but not analyzed for mutants. Collected tissues were weighed, flash frozen in liquid nitrogen, transferred to a freezer box on dry ice, and then stored in a freezer set at or below 60°C. Liver and glandular stomach tissue samples from the first five animals/group in Groups 1, 2, 3, 5 and 6 were processed for DNA isolation and analysis of cII mutants, following BioReliance SOPs.

No mortality was noted in this study. During the 5-day range-finding assay, a lack of weight gain was noted in the 750 mg/kg/day dose group. For the mutation assay cohort, decrease motor activity was noted occasionally in Oxirane-treated animals, at all dose levels. In addition, mean total body weight gains (Day 1-31) were significantly lower (37.2%) as compared to the vehicle control, at the 750 mg/kg/day dose level.

Treatment with Oxirane at dose levels up to 750 mg/kg/day did not cause biologically significant increases in mutant frequency (MF) at the cII gene in the liver or glandular stomach of Big Blue® male rats. Although a statistically significant increase in MF (1.7-fold as compared to the vehicle control) was noted at 750 mg/kg/day for glandular stomach, this value was slightly lower than the historical background mean (30.3 x 10-6 vs. 34.2 x 10-6 respectively). The positive control treatment with ENU produced statistically significant increases in MF for all tissues tested, demonstrating the utility of the test system to detect and quantify induced mutants, following exposure to a known direct acting mutagen. The study design and results obtained met protocol-specified assay acceptance criteria and were consistent with the study requirements of OECD TG 488 for transgenic rodent mutation assays.

A total of 27 samples were analyzed for the presence of glycidyl lauryl ether (GLE). Although several samples had detectable levels of GLE, no sample had results greater than the LLOQ of the method (2 μg/mL plasma). The initial LLOQ of the method was 1 μg/mL plasma, but due to the failure of the lowest calibration standard, the LLOQ of the method was raised to 2 μg/mL plasma.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Gene mutation on bacteria

Two reverse mutation tests on bacteria (on S. typhimurium and on E. coli) are available and valid. Both tests have been realised according to protocol similar to the OECD guidelines. Results are ambiguous.

The first study was realised according to the OECD 471 guideline and in compliance with GLP, using the direct plate incorporation method in S. typhimurium TA98, TA 1535, TA1537, TA100 strains, at doses of 3.3, 10, 33, 100, 333, 1000, 3333µg/plate (and at higher concentrations during the preliminary test) (Valentine, 1997).In the mutagenicity assay, a positive response was observed with tester strain TA1535, with and without metabolic activation. All other strains were negative, with and without metabolic activation. 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, alkyl glycidyl ether was concluded to be positive in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay.

In the second test, S. typhimurium strain TA1535, TA1537, TA1538, TA98 and TA100 were tested for mutagenic activity in a protocol similar to the OECD 471 guideline, at doses of 0.6µg/plate, 1.85µg/plate, 5.5µg/plate, 16.7µg/plate, 50µg/plate and 150µg/plate with and without metabolic activation (McCarrol, 1979). Results indicate that a limited response of strain TA1535 ranging from 3 to 4 fold at 50 and 150 µg/plate was produced by metabolism of the test agent to a weakly active form. About a two-fold increase in revertants of TA100 was also obtained at the same dose levels. Strain TA98 responded in a similar manner, yielding approx. two to three-fold increases in revertant numbers at all test concentrations in the non-activated test. Results were found to be ambiguous in all strains.

In vitro gene mutation on mammalian cells:

Two gene mutation tests on mammalian cells are valid available (one CHO/HGPRT and oneL5178Y/TK).Both tests have been realised according to protocol similar to the OECD guidelines and give homogeneous negative results.

In the first study, alkyl glycidyl ether was tested in the CHO/HGPRT Mutation Assay in the absence and presence of metabolic activation, according to the OECD 476 guideline and in compliance with GLP (Richard, 1998). Without S9 activation, no cultures exhibited mutant frequencies of >40 mutants per 10e6 clonable cells. With S9 activation, the mutant frequency of the highest dose seeded for mutant selection was elevated, though it was not >40 mutants per 10e6 clonable cells; in the independent repeat assay the high dose exhibited a mutant frequency> 40 mutants per 10e6 clonable cells; in the third assay, no cultures exhibited mutant frequencies of >40 mutants per 10e6 clonable cells. This indicates that the high mutant frequency exhibited in the independent repeat with S9 activation was probably an artifact. There was no dose response in any of the mutagenesis assays. Therefore, the results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, alkyl glycidyl ether was concluded to be negative with and without S9 activation.

In the second studyrealized according to a protocol similar to the OECD 476, mouse lymphoma L5178Y cells were treated withalkyl glycidyl etherat doses of 31.75µg/mL, 62.5µg/mL, 125µg/mL, 250µg/mL and 500µg/mL with and without metabolic activation (Piper, 1979a). Underthe experimental conditions alkyl glycidyl ether did not showed any evidence of mutagenicity.

In vivomicronucleus test:

Two different studies are available and valid and both results are consistent and indicate an absence of mutagenicity.

The first study was realised according to the OECD 474 guideline and in compliance with GLP (Ramadevi, 1997). Male and female mice were dosed with alkyl glycidyl ether at concentrations of 1000, 2000 or 4000 mg/kg body weight. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs following dose administration included: lethargy in male and female mice at all test article dose levels and piloerection in male and female mice at 4000 mg/kg. Bone marrow cells, collected 24, 48 and 72 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 11%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the treated groups relative to the respective vehicle controls. No significant increase in micronucleated polychromatic erythrocytes in treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hours after dose administration. The results of the assay indicate that under the conditions described in this report, alkyl glycidyl ether did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. Alkyl glycidyl ether was concluded to be negative in the mouse micronucleus assay.

The second study supports those negative results (Pullin, 1977). The test was realised on female mice treated orally with alkyl glycidyl ether at 1000mg/kg/d during 5 days. Micronuclei were then evaluated in the bone marrow. Alkyl glycidyl ether gave almost identical micronuclei values as the control and thus this chemical did not produce a mutagenic effect in terms of micronuclei at the dosage tested.

In vivochromosomal aberration test:

One study is available and valid. In this study, realised according to the OECD 475 guideline, alkyl glycidyl ether was administered intraperitoneally for five consecutive days at concentration as high as 712.5 mg/kg/day in male and female Sprague Dawley rats (Piper, 1979b). Micronuclei were then evaluated in bone marrow cells collected from both femurs. Under the conditions of this study, no positive result in any consistent structural or numerical cytogenetic aberrations in rat bone marrow cells was observed. Alkyl glycidyl ether was therefore negative in this assay.

Conclusion

Although C12-C14 alcohol MGEproduced an ambiguous response in the gene mutation test on S. typhimurium, results of the gene mutation tests on mammalian cells and of thein vivomicronucleus tests in mice and of thein vivochromosomal aberration assay in rats gave a negative response.C12-C14 alcohol MGE is therefore considered as negative for mutagenicity.


Short description of key information:
Although Oxirane, 2-((C12-14-alkyloxy)methyl)derivs produced an ambiguous response in the gene mutation test on S. typhimurium. Further in vivo testing was used to clarify it's genotoxicity potential. Results of the gene mutation tests on mammalian cells and of the in vivo micronucleus tests in mice, chromosomal aberration assay in rats and a Transgenic Rodent Assay gave a negative response. Oxirane, 2-((C12-14-alkyloxy)methyl)derivs is therefore considered as negative for mutagenicity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In vivo testing, conducted to relevant OECD guidelines and under GLP certification, was used to clarify genotoxic potential. Results of the gene mutation tests on mammalian cells and of the in vivo micronucleus tests in mice, chromosomal aberration assay in rats and a Transgenic Rodent Assay gave a negative response. Oxirane, 2-((C12-14-alkyloxy)methyl)derivs is therefore considered as negative for mutagenicity.

Oxirane, 2-((C12-14-alkyloxy)methyl)derivs is not genotoxic in vivo, Classification and Labeling as a Mutagen is not required.