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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 471, EPA OPPTS 870.5100, EU method B13/14 and in accordance with the Principles of Good Laboratory Practices (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:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Some minor exceptions were noted, however these did not have any impact on the outcome or results of the 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-butoxyethyl benzoate
EC Number:
226-685-8
EC Name:
2-butoxyethyl benzoate
Cas Number:
5451-76-3
Molecular formula:
C13H18O3
IUPAC Name:
2-butoxyethyl benzoate
Test material form:
other: variable coloured liquid
Details on test material:
- Name of test material (as cited in study report): Butyl Cellosolve™ Benzoate
- Physical state: variable coloured liquid
- Analytical purity: 99+%
- Lot/batch No.: 02112012-JLT
- Expiration date of the lot/batch: 11 February 2013
- Storage condition of test material: Ambient (+18 to +36 ºC)

Method

Target gene:
The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.
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):
- Type and identity of media: 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.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: The growth requirements and the genetic identity of strains like histidine or tryptophan requirement, sensitivity to UV radiation, resistance of strains TA98, TA100 and WP2uvrA (pKM101) to ampicillin and rfa mutation of Salmonella typhimurium strains were checked along with the range of spontaneous revertants after preparation of the master plates.
- Periodically "cleansed" against high spontaneous background: yes
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:
Initial Toxicity mutation Assay - G2 - 1.5 µg, G3 - 5 µg, G4 - 15 µg, G5 - 50 µg, G6 - 150 µg, G7 - 500 µg, G8 - 1500 µg and G9 - 5000 µg
Confirmatory Mutation Assay - G2 - 32 µg, G3 - 101 µg, G4 - 320 µg, G5 - 1013 µg, G6 - 3200 µg, G7 - 50 µg, G8 - 158 µg, G9 - 500 µg, G10 - 1580 µg and G11 - 5000 µg
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: recommended vehicle by various regulatory agencies
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
Performance of the Assay -
Labeling - The petridishes were labelled to indicate the study number, strain number, treatment group, test phase and activation.
Number of Replicates - Two replicate plates were maintained for the initial toxicity-mutation assay and three replicate plates were maintained for the confirmatory mutation assay.
Plating Procedure - 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.
A. Presence of metabolic activation -
a) 100 μl test dose/vehicle/appropriate positive control
b) 100 μl bacterial culture
c) 500 μl S-9 mix
B. Absence of metabolic activation
a) 100 μl test dose/vehicle/appropriate positive control
b) 100 μl bacterial culture
c) 500 μl of PBS
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.
Viable Counts - 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
Evaluation criteria:
The conditions necessary for determining a positive result were - there should be 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.
For strains TA98, TA1535, and TA1537 - Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.
For strain TA100 and WP2uvrA (pKM101) - Data sets were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2.0-times the mean vehicle control value.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) were not considered as positive.
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
S-9 Homogenate
a) Sterility Check - The S-9 homogenate was found to be sterile.
b) Metabolic Activation - The S-9 homogenate was found to be active.
c) Protein Content - The protein content of the S-9 homogenate was 27.5 mg/mL.
Genotypic Characterization - Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 demonstrated the requirement of histidine amino acid for their growth. Escherichia coli strain WP2uvrA (pKM101) demonstrated the requirement of tryptophan amino acid for its growth. Ampicillin resistance was demonstrated by the strains TA98, TA100 and WP2uvrA (pKM101) which carry R-factor plasmids. The presence of characteristic mutations like the rfa mutation was demonstrated by all the Salmonella typhimurium strains by their sensitivity to crystal violet. The uvrA mutation in the Escherichia coli strain and the uvrB mutation in the Salmonella typhimurium strains were demonstrated through their sensitivity to ultraviolet light.
Finally, all these tester strains produced spontaneous revertant colonies which were within the frequency ranges of the test facility’s historical control data.
Viable Counts of the Overnight Culture - 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.
Stability Analysis of Dose Formulation Samples - The test substance was stable in DMSO at 15 and 50000 μg/mL after 4 and 24 hours.
Concentration Analysis of Dose Formulations - The results of the concentration analysis for the initial toxicity-mutation assay indicated that the actual mean concentrations of the analyzed dose levels were between 89.5 and 105 % of their respective nominal target concentrations confirming that the concentration of the test substance was within acceptable limits (85 to 115 % of nominal concentrations and an RSD of < 10 %), except for the dose 15 μg/mL, which was higher than the acceptable limit (117 %), no additional testing was conducted to investigate this finding. This indicates that the regulatory-required top dose level (5000 μg/plate) was achieved. No test substance was detected in the vehicle control.
The results of the concentration analysis for the confirmatory mutation assay indicated that the actual mean concentrations of the analyzed dose levels were between 101 and 109 % of their respective nominal target concentrations confirming that the concentration of the test substance was within acceptable limits (85 to 115 % of nominal concentrations and an RSD of < 10 %), except for the dose 500 μg/mL, which was higher than the acceptable limit (180 %), no additional testing was conducted to investigate this finding. This indicated that the regulatory-required top dose level (5000 μg/plate) was achieved. No test substance was detected in the vehicle control.
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 on the basal agar plates at any of the tested doses
but showed differential toxicity profile. No toxicity was observed in any of the tester strains up to 500 µg/plate, either in the presence or absence of metabolic activation when compared to the vehicle control. However, for TA98, TA100, TA1535 and TA1537, there was a slight reduction in the intensity of bacterial background lawn at 1500 µg/plate and moderate reduction at 5000 µg/plate. For WP2uvrA (pKM101), there was a slight reduction in the intensity of bacterial background lawn at 1500 as well as at 5000 µg/plate. There was no positive mutagenic response observed in any of the strains in any of the tested doses either in the presence or in the absence of 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.
Confirmatory 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 on the basal agar plates at any of the tested
doses. No toxicity was observed in the tester strains TA98, TA100, TA1535 and TA1537 up to 320 µg/plate and up to 500 µg/plate in the strain WP2uvrA (pKM101), either in the presence or absence of metabolic activation when compared to the vehicle control. However, for TA98, TA100, TA1535 and TA1537, there was a slight reduction in the intensity of bacterial background lawn at 1013 and 3200 µg/plate. For WP2uvrA (pKM101), there was a slight reduction in the intensity of bacterial background lawn at 1580 as well as at 5000 µg/plate. There was no positive mutagenic response observed in any of the strains in any of the tested doses either in the presence or in the absence of 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.
Sterility Controls - The most concentrated test substance dilution, the Sham (PBS) and S9 mixes were found to be sterile.
Both the Salmonella typhimurium and Escherichia coli tester strains were found to be reliable and responsive to the different genotypic characterization tests, e.g., amino acid requirement, rfa mutation, uvr mutation and the R-factor plasmids. Similarly, the spontaneous revertant counts of the vehicle control groups of these tester strains were within the ranges of the test facility’s historical control data.
The positive controls produced a more than 3-fold increase in the mean numbers of revertant colonies when compared to the respective vehicle controls, demonstrating the sensitivity of the assay procedure.
The test substance, Butyl CELLOSOLVE™ Benzoate tested at doses up to 5000 μg/plate, did not show any positive mutagenic response in any of the tester strains in any of the tested doses either in the presence or absence of metabolic activation when compared to 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

Under the conditions of this assay, it is concluded that the test substance, 2-butoxyethyl benzoate was negative (non-mutagenic) in this Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay.
Executive summary:

In a confirmatory mutation assay, 2 -butoxyethyl benzoate was exposed in triplicate at 32, 101, 320, 1013 and 3200 µg/plate test doses with TA98, TA100, TA1535 and TA1537 and at 50, 158, 500, 1580 and 5000 µg/plate test doses with WP2uvrA (pKM101) along with the vehicle and appropriate positive controls. The mean and standard deviation of revertant colonies were calculated for each test dose and the controls for all the tester strains. The test substance did not precipitate on the basal agar plates at any of the tested doses. No toxicity was observed in the tester strains TA98, TA100, TA1535 and TA1537 up to 320 µg/plate and up to 500µg/plate in the strain WP2uvrA (pKM101), either in the presence or absence of metabolic activation when compared to the vehicle control. However, for TA98, TA100, TA1535 and TA1537, there was a slight reduction in the intensity of bacterial background lawn at 1013 and 3200 µg/plate. For WP2uvrA (pKM101), there was a slight reduction in the intensity of bacterial background lawn at 1580 as well as at 5000 µg/plate. There was no positive mutagenic response observed in any of the strains in any of the tested doses either in the presence or in the 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 results of concentration analysis of dose formulations from the initial and confirmatory mutation assays confirmed that the regulatory-required top dose level (3200 and 5000 μg/plate) was achieved in both assays and the results support the validity of the study conclusion. Under the conditions of the current study, the test substance, 2 -butoxyethyl benzoate was negative (nonmutagenic) in thisSalmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay.