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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
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted as per OECD TG 471, US EPA OPPTS 870.5100, EC: B.13/14, METI and in accordance with the Principles of Good Laboratory Practice (GLP).
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Exception: Concentration checks were not performed for the positive control substances (2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine, 4-nitroquinoline-1oxide, and sodium azide) used in this study.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Remarks:
same as above
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Remarks:
same as above
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(2-phenoxyethoxy)ethanol
EC Number:
203-227-5
EC Name:
2-(2-phenoxyethoxy)ethanol
Cas Number:
104-68-7
Molecular formula:
C10H14O3
IUPAC Name:
2-(2-phenoxyethoxy)ethanol
Details on test material:
- Name of test material (as cited in study report): Diethylene glycol mono phenyl ether
- Physical state: colourless to yellow liquid
- Analytical purity: 99.92%
- Lot/batch No.: 201103261-7-18
- Storage condition of test material: Ambient (+18 to +36 ºC)

Method

Target gene:
Histidine auxotrophic strains of Salmonella typhimurium viz., TA98, TA100, TA1535, and TA1537 and Tryptophan auxotrophic strain of Escherichia coli WP2uvrA (pKM101) were used in the study.
Each S. typhimurium tester strain contained, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation resulted in a cell wall deficiency that increased the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene resulted in a deficient DNA excision-repair system. Tester strains TA98 and TA100 also contained the pKM101 plasmid (carrying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.
TA98 and TA1537 were reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions.
The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene. Tester strain WP2uvrA (pKM101) has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base substitution mutations.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 homogenate was used as the metabolic activation system.
Test concentrations with justification for top dose:
In the initial toxicity-mutation assay, diethylene glycol mono phenyl ether was exposed in duplicate at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate test doses along with the vehicle and appropriate positive controls. In the confirmatory mutation assay, diethylene glycol mono phenyl ether was exposed in triplicate at 100, 266, 707, 1880 and 5000 μg/plate test doses along with the vehicle and appropriate positive controls
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Diethylene glycol mono phenyl ether was found to be insoluble in sterile water (SW) at 50 mg/mL and soluble in dimethyl sulfoxide (DMSO) at the required concentration of 500 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Nitrofluorene (Batch no.S08447-476), 2-Aminoanthracene Batch no. (STBB1901), 9-Aminoacridine (Batch no. 106F06681), 4-Nitroquinoline-1-Oxide (Batch no. 067K1267) and Sodium azide (Batch no. 098K0052)
Remarks:
All positive controls were either sourced from Sigma-Aldrich, Germany or Sigma Chemical Co., USA or Sigma-Aldrich, Inc., USA
Details on test system and experimental conditions:
The tester strains of Salmonella typhimurium was sourced from Health Protection Agency, National Collection of Type, Cultures (NCTC) 61, Colindale Avenue, London NW9 5EQ, Great Britain and Escherichia coli was sourced from The National Collection of Industrial and Marine Bacteria Ltd. (NCIMB),
Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, U.K.

Stock cultures of tester strains were stored in Oxoid Nutrient Broth No. 2 in the test facility as frozen permanents in liquid nitrogen. Laboratory stocks were maintained on respective minimal glucose agar plates as master plates of each strain, for a maximum period of 2 months and refrigerated at 2 to 8 ºC.

S-9 homogenate was prepared in batches and stored in a deep freezer maintained at
-68 to -86 ºC.

S-9 mix was prepared by mixing one part of S-9 homogenate with 9 parts of co-factors (NADP, glucose-6-phosphate, magnesium chloride and potassium chloride) solution. The co-factors solution was prepared by dissolving the following in 50 mL of cold PBS (pH 7.4) and filter sterilized using a 0.2 µm disposable syringe filter. S-9 mix was prepared by mixing 5.5 mL of the S-9 homogenate with 49.5 mL of the co-factors solution, kept in an ice bath and used within one hour.

Stability of dose formulations of the test substance at 15.2 and 185004 μg/mL was established in DMSO after 4 hours at room temperature using a validated analytical method.

Each of the tester strains from the master plates were inoculated into the respective tubes containing Oxoid Nutrient Broth No. 2 and the tubes were incubated at 37 ±1 °C for 17 hours.

Initial Toxicity Mutation Assay - A quantity of 500 mg of the test substance was dissolved in DMSO and the volume was made up to 10 mL with DMSO in a volumetric flask (50000 µg/mL). This stock solution was further diluted in DMSO to prepare the 8 test substance concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Confirmation Mutation Assay - A quantity of 1250 mg of the test substance was dissolved in DMSO and the volume was made up to 25 mL with DMSO in a volumetric flask (50000 µg/mL). This stock was further diluted in DMSO as follows to prepare 5 test substance concentrations of 100, 266, 707, 1880 and 5000 μg/plate.
Two replicate plates were maintained for the initial toxicity-mutation assay and three replicate plates were maintained for the confirmatory mutation assay.
The initial toxicity-mutation, as well as the confirmatory mutation assay was conducted using the pre-incubation assay method at ambient temperature under yellow light. These test constituents were transferred into sterile test tubes and were kept in an incubator shaker for approximately 20 ± 2 minutes at 37 ± 1 ºC. After this period, 2 mL of soft agar containing histidine-biotin / tryptophan was added to each of the tubes and the constituents were overlaid onto VB agar plates. After the soft agar had set, the plates were incubated at 37 ± 1 °C for 67 hours. After incubation, the revertant colonies in each plate were counted manually and the plates were examined for bacterial background lawn. The bacterial suspension of each tester strain was diluted up to 10-6 dilution in PBS. One hundred microliters (100 μL) from the 10-6 dilution of each tester strain was plated onto nutrient agar plates in triplicate. The plates were incubated at 37 ± 1 °C for 67 hours. After incubation, the number of colonies in each plate were manually counted and expressed as the number of colony forming units per mL of the bacterial suspension. Revertant colonies of all the tester strains for the controls and each test concentration were counted manually. Sterility control plates were observed for microbial colonies. Colonies of all the tester strains on nutrient agar plates were counted manually.
Evaluation criteria:
A dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test substance either in the absence or presence of the metabolic activation system.
Statistics:
Standard statistical methods were employed

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
The S-9 homogenate was found to be sterile and active. The protein content of the S-9 homogenate was found to be 26.6 mg/mL.
Genotypic characterization was done for all the tester strains and they produced spontaneous revertant colonies which were within the frequency ranges of the historical control data. Viable counts of all the tester strains were within the required range of 1-2x109 CFU/mL for the initial toxicity-mutation as well as for the confirmatory mutation assay. The test substance was stable after 4 hours in DMSO at 15.2 and 185004 μg/mL.
Initial Toxicity – Mutation Assay - The mean number of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains. The test substance did not precipitate in the basal agar plates at any of the tested doses, either in the presence or in the absence of metabolic activation. No toxicity was observed as the intensity of the bacterial background lawn was comparable to the vehicle control up to 5000 µg/plate, in the presence and absence of metabolic activation.
The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for all tester strains when compared to the respective vehicle control plates, either in the presence or absence of the metabolic activation. Positive control chemicals tested simultaneously produced more than a 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.
Confirmation Mutation Assay - The mean number of revertant colonies/plate in the DMSO control was within the range of the in-house spontaneous revertant counts for all the tester strains.
The test substance did not precipitate in the basal agar plates at any of the tested doses, either in the presence or in the absence of metabolic activation. No toxicity was observed as the intensity of the bacterial background lawn was comparable to the vehicle control up to 5000 µg/plate, in the presence and absence of metabolic activation.
The tested doses showed no positive mutagenic increase in the mean number of revertant colonies for all tester strains when compared to the respective vehicle control plates, either in the presence or absence of the metabolic activation. Positive control chemicals tested simultaneously produced more than a 3-fold increase in the mean numbers of revertant colonies for all the strains when compared to the respective vehicle control plates. No toxicity was observed in the positive controls as the intensity of the bacterial background lawn of all the tester strains was comparable to that of the respective vehicle control plates.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results from the initial and confirmatory assays, indicate the tested doses showed no positive mutagenic increase in the mean numbers of revertant colonies for all tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation. The study indicated that the test substance, diethylene glycol mono phenyl ether, was negative (non-mutagenic) in this Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay at the doses tested and the conditions of testing employed.
Executive summary:

Diethylene glycol mono phenyl ether was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM 101) strain of Escherichia coli in two phases. In the first phase, an initial toxicity-mutation test was performed. The second phase was an independent confirmatory mutation test. The bacterial tester strains were exposed to the test substance in the presence and absence of a metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver) using a pre-incubation procedure.

Diethylene glycol mono phenyl ether was found to be insoluble in sterile water (SW) at 50 mg/mL and soluble in dimethyl sulfoxide (DMSO) at the required concentration of 500 mg/mL. Diethylene glycol mono phenyl ether was stable in DMSO at 15.2 and 185004 μg/mL after 4 hours.

In the initial toxicity-mutation assay, diethylene glycol mono phenyl ether was exposed in duplicate at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate test doses along with the vehicle and appropriate positive controls. In the confirmatory mutation assay, diethylene glycol mono phenyl ether was exposed in triplicate at 100, 266, 707, 1880 and 5000 μg/plate test doses along with the vehicle and appropriate positive controls.

The test substance did not cause any precipitation on the basal agar plates. No toxicity was observed as the intensity of the bacterial background lawn was comparable to the vehicle control up to 5000 μg/plate, in the presence and absence of metabolic activation. The mean and standard deviation of revertant colonies were calculated for each test dose and the controls for all the tester strains.

The results from the initial and confirmatory assays, indicate the tested doses showed no positive mutagenic increase in the mean numbers of revertant colonies for all tester strains when compared to the respective vehicle control plates, either in the presence or absence of metabolic activation.

In this study, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay.

The analytically-determined concentrations of diethylene glycol mono phenyl ether in the initial assay dose formulations ranged from 76.7 to 100.1 % of their respective targets concentrations. Although the analysis results of the lowest concentration (15 μg/mL) was not within the specified limit, all remaining dose levels, including the critical top concentration, were within the acceptable range of 85 to 115 % of target and < 10 % RSD. The results of the concentration analysis for the confirmatory mutation assay were within acceptable limits, as the actual mean concentrations were between 89.6 and 101.2 % of their respective nominal target concentrations. The regulatory-required top dose level (5000 μg/plate) was achieved in both assay and the results support the validity of the study conclusion. No test  substance was detected in the vehicle control.

All criteria for a valid study were met as described in the protocol. The study indicated that the test substance, diethylene glycol mono phenyl ether, was negative (non-mutagenic) in this Salmonella- Escherichia coli/Mammalian-Microsome Reverse Mutation Assay at the doses tested and the conditions of testing employed