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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-10-14 to 2013-10-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
Benzeneacetaldehyde, cyclic acetal with glycerol
EC Number:
249-934-2
EC Name:
Benzeneacetaldehyde, cyclic acetal with glycerol
Cas Number:
29895-73-6
Molecular formula:
C11H14O3
IUPAC Name:
Reaction mass of cis-2-benzyl-1,3-dioxan-5-ol and rel-[(2R,4R)-2-benzyl-1,3-dioxolan-4-yl]methanol and rel-[(2R,4S)-2-benzyl-1,3-dioxolan-4-yl]methanol and trans-2-benzyl-1,3-dioxan-5-ol
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- E. Coli:Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
- Initial Toxicity-Mutation Assay:
TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA (without and with S9): dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate.
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 150, 500, 1500 or at 5000 μg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum doses plated in the confirmatory mutagenicity assay were 1500 μg per plate with all Salmonella tester strains in the absence of S9 activation and 5000 μg per plate for all other strains and test conditions.

- Confirmatory Mutagenicity Assay:
The following dose levels were used:
TA 1535, TA 1537, TA 98, TA 100 (without S9): 1.5, 5.0, 15, 50, 150, 500 and 1500 µg/plate
TA 1535, TA 1537, TA 98, TA 100 (with S9) and E. coli WP2 uvrA (with and without S9): 5.0, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL, the maximum concentration tested in the solubility test conducted at BioReliance.
Controls
Untreated negative controls:
other: Sterility control
Remarks:
(Vehicle control, test substance dilutions, S9 and Sham mixes)
Negative solvent / vehicle controls:
yes
Remarks:
(50 µL/plate DMSO)
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Initial Toxicity Mutation Assay: in agar (pre-incubation method)
- Confirmatory Mutagenicity Assay: in agar (pre-incubation method)

DURATION
- Exposure duration:
pre-incubation 60 ± 2 minutes
incubation 48 - 72 hours

NUMBER OF REPLICATIONS:
- Initial Toxicity Mutation Assay: Doses of the test substance, vehicle control and positive controls were tested in duplicate.
- Confirmatory Mutagenicity Assay: Doses of the test substance, vehicle control and positive controls were tested in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. The condition of the background lawn was scored based on characteristics of a thinning of the microcolony lawn and increase in size of microcolonies compared to the vehicle control plate. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn.
Evaluation criteria:
EVALUATION OF RESULTS:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.

Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative, if it was neither positive nor equivocal.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
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:
other: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
other: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

INITIAL TOXICITY-MUTATION ASSAY:
- Toxicity was observed beginning at 150, 500, 1500 or at 5000 µg/plate in the initial toxicity-mutation assay.

COMPARISON WITH CONTROL DATA:
- The vehicle controls were within the characteristic mean number of spontaneaus revertants of the cultures.
- The mean of each positive control showed at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity was observed beginning at 500, 1500 or at 5000 µg/plate in the confirmatory mutagenicity assay.

VALIDITY:
All criteria for a valid study were met as described in the protocol.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the substance is not mutagenic in the Bacterial Reverse Mutation Assay performed according to OECD 471 (1997).
Executive summary:

The mutagenic activity of the substance was evaluated in the Bacterial Reverse Mutation Assay in accordance with OECD 471 (1997) and GLP principles. The test was performed in two independent experiments, using the preincubation method, using S. thyphimurium tester strains TA1535, TA1537, TA98, TA100 and E. coli tester strain WP2 uvrA, in the absence and presence of Aroclor-induced rat liver S9. In the initial toxicity-mutation assay dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Toxicity was observed beginning at 150, 500, 1500 or at 5000 μg per plate. The maximum dose levels used in the confirmatory mutagenicity test were selected based on the findings of the initial toxicity-mutation assay. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500 and 1500 μg per plate with all Salmonella tester strains in the absence of S9 activation and 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate with the remaining strains and test conditions. Toxicity was observed beginning at 500, 1500 or at 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Adequate vehicle and positive controls were included. All criteria for a valid study were met. The results indicate that, under the conditions of this study, the substance did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of S9 activation. Under the conditions of this study, test substance was concluded to be negative in the Bacterial Reverse Mutation Assay.