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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:
1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study methodology followed was equivalent or similar to OECD TG 471 and in accordance with the Principles of Good Laboratory Practices (GLP)
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Cyclohexane dimethylol diglycidyl ether
- Analytical purity: 53.7% by GC
- Ref. No.: 27673-9

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 / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Top Agar
- Properly maintained: yes
- Spot marker tests were used to characterize the bacteria were run periodically (approximately at 4-6 months interval) to confirm the genetic composition
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
- Type and identity of media: Top Agar
- Properly maintained: yes
- Spot marker tests were used to characterize the bacteria were run periodically (approximately at 4-6 months interval) to confirm the genetic composition
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate
Test concentrations with justification for top dose:
The cyclohexane dimethylol diglycidyl ether-containing mixture was prepared at concentrations of 100, 10, 1, 0.1, and 0.01 mg per ml in DMSO. Since 0.1 ml of the test chemical solution was added per culture, the final amounts tested were 10, 1, 0.1, 0.01, and 0.001 mg of test material per plate. In a repeat assay, 10, 1, 0.1, and 0.01 mg of the test material per plate were evaluated.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: recommended by various regulatory agencies
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
2-nitrofluorene
other: N-methyl-N'-nitro-N-nitrosoguanidine, Quinacrine mustard 2HCl, 2-anthramine, 8-aminoquinoline
Details on test system and experimental conditions:
The top agar consisted of 0.6% Bacto-Difco agar and 0.5% NaCl. Each 100 ml of top agar was supplemented with biotin and a trace of histidine (10 ml of 0.5 mM L-histidine HCl and 0.5 mM biotin). A trace of histidine in the top agar allowed the bacteria on the plate to undergo several replications which is necessary for observing mutagenesis. The petri plates contained 30 ml of minimal-glucose agar medium (1.5% Bacto-Difco agar in Vogel-Bonner Medium E with 2% glucose).
The preincubation assay was employed t o optimize the contact between the bacteria and test chemical. The preincubation assay (a modification of the standard plate assay) in which the bacteria, test chemical and, if necessary, an activation mixture were incubated in closed vials for 30 min. at 30°C in a shaking water bath incubator. After the 30 min. incubation, 2 ml of top agar were added and the plates poured and placed in a 37°C incubator for 48 hr. All negative and positive controls were also preincubated. The number of revertant colonies per plate were counted with an Artek counter (Model 880, Artek Systems Corp., Farmingdale NY). The total volume of incubation mixture was 0.8 ml and included:
1. 0.5 ml of S-9 activation mixture (0.1 ml for TA1537) or 0.5 ml of Ames buffer.
2. 0.1 ml of Ames buffer (0.5 ml for TA1537)
3. 0.1 ml of an overnight culture
4. 0.1 ml of test chemical solution
where Ames buffer is defined per ml as, MgCl2 (8 µmoles), KCl (33 µmoles) and sodium phosphate dibasic pH 7.4 (100 µmoles), and the S-9 activation mixture is 5 ml of S-9 rat liver homogenate in 20 ml of Ames buffer supplemented with (per ml) 5 µmoles glucose-6-phosphate and 4 µmoles NADP.
Evaluation criteria:
A test chemical was considered a bacterial mutagen if the mean number of revertant colonies was at least three times higher than the mean of the negative (solvent) control and it produced a dose response relationship over several concentrations. If a chemical produced reproducible reversion rates in excess of 3X over background but no definitive dose-response relationship it was considered to be a presumptive bacterial mutagen. If a chemical produced reproducible reversion rates greater than 2X but less than 3X over controls the results were considered equivocal or inconclusive.
Statistics:
Standard statistical methods were employed

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity (no growth on plates or a sparse background lawn) was observed in both assays in all strains tested without activation at 10 mg test material per plate.
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material was tested with and without metabolic activation at 10, 1, 0.1, 0.01, and 0.001 mg per plate in strains TA98, TA1535, TA1537, and TA1538 in an initial assay and at 10, 1, 0.1, and 0.01 mg per plate in strains TA98, TA100, TA1535, TA1537, and TA1538 in a second assay. Toxicity (no growth on plates or a sparse background lawn) was observed in both assays in all strains tested without activation at 10 mg test material per plate.
Test chemical toxicity often permits non-revertants to grow as more histidine becomes available to individual surviving bacteria. Replica plating (transfer to histidine deficient plates ) may be used to differentiate between true revertant colonies and non-revertant (overgrown background) colonies. Accordingly, the presumed revertants observed at 10 mg per plate in strain TA1537 with activation were confirmed to be non-revertants by replica plating (the colonies did not grow when transferred to histidine deficient plates) . Apparent genetic activity was observed in strain TA1535 without metabolic activation at 1 mg/plate and in TA1535 with activation at 0.1, 1 and 10 mg/plate. Replica plating of TA1535 at 1 mg per plate non-activated and 0.1 mg per plate activated confirmed that the colonies observed were true revertants (the colonies grew when transferred to histidine deficient plates).
In the repeat study, the cyclohexane dimethylol diglycidyl ether-containing mixture was again active in strain TA1535. In addition, the test material was also found to be mutagenic in strain TA100 without activation at 1 mg/plate and with activation at 1 and 10 mg/plate. Due to technical difficulties TA100 had not been evaluated in the first assay. The suggested positive response in strain TA1537 without metabolic activation at 0.1 mg/plate in the first assay was not repeatable in the second assay. Thus, the cyclohexane dimethylol diglycidyl ether-containing mixture was found to produce a dose-related mutagenic response only in strains TA100 and TA1535, both strains which are primarily sensitive to chemicals producing base-pair substitutions mutations.
The activity of the positive controls demonstrated the responsiveness of the bacteria to known mutagens in the present evaluation. Comparison of the test material treated plates to the negative controls (spontaneous reversion) indicates that a positive response (3-fold increase over controls and a dose response) was elicited in strains TA100 and TA1535, both with and without the addition of the mammalian metabolic activation preparation. While it is conceivable that the observed positive response may be due to a contaminate since the test substance contained only 53.7% cyclohexane dimethylol diglycidyl ether, the bacterial mutagenic activity of the parent compound must be presumed until shown otherwise with a purer sample. It is therefore concluded that cyclohexane dimethylol diglycidyl ether be considered a base-pair substitution mutagen in this highly sensitive bacterial mutagenicity assay.
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):
positive

Under the conditions of the study, the test material cyclohexane dimethylol diglycidyl ether is considered a base-pair substitution mutagen.
Executive summary:

A cyclohexane dimethylol diglycidyl ether-containing mixture was evaluated for genetic activity in the AMES preincubation/plate incorporation bacterial mutagenicity assay, with and without the addition of a mammalian metabolic activation preparation. The study was conducted using Salmonella typhimurium bacterial tester strains TA98, TA100, TA1535, TA1537 and TA1538. The test material was tested in all strains at 10, 1.0, 0.1, 0.01 and 0.001 (except TA100) mg per plate. Toxicity was observed at 10 mg/plate in all strains without metabolic activation. The test material was found to produce a dose-related mutagenic response in strains TA100 and TA1535, strains which are sensitive to base-pair substitution mutagens. The observed mutagenic response in strains TA100 and TA1535 was markedly enhanced by the presence of the mammalian metabolic activation preparations. Thus, cyclohexane dimethylol diglycidyl ether is considered to be a bacterial mutagen of the base-pair substitution type when evaluated in this highly sensitive bacterial mutagenicity assay.