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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:
weight of evidence
Study period:
15 June 1992 - 03 July 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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): Di-Trimethylolpropane, Di-TMP
- Lot/batch No.: P920145-3
- Storage condition of test material: At room temperature in the dark.
- Other: Test substacne characterization (purity, stability etc.) was the responsibility of the Sponsor.

Method

Target gene:
Reversion to histidine independence
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction supplemented with salts and co-factors (S9-mix)
Test concentrations with justification for top dose:
Preliminary test - a threefold dilution series from 5.0 to 0.062 mg/plate.
Main test (mutagenicity test) - 5.0, 2.5, 1.3, 0.63, 0.31 mg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: initial investigations found that the test substance was readily soluble in water up to at least 16.7 mg/mL.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Used for strains TA100 and TA1535 in the absence of S9- mix. Migrated to IUCLID6: 1.0µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For strains TA98 and TA 1537 in the absence of S-9 mix. Migrated to IUCLID6: 1.0 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthrazene, 1.5µg/plate
Remarks:
For all four strains in the presence of S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: First test series: in agar (plate incorporation);
Second test series: preincubation


DURATION
- Preincubation period: 30 minutes (second test series only)
- Exposure duration: 48 to 72 hours



NUMBER OF REPLICATIONS: Three plates per dose level.


DETERMINATION OF CYTOTOXICITY
- Method: Depression of the background growth.


OTHER EXAMINATIONS:
- Other: Observation of the number of revertant colonies.


OTHER:
Evaluation criteria:
The data obtained were evaluated with respect to the following criteria:
- Dose-response relationship.
- A doubling or more of the revertant level as compared to the control level.
Statistics:
Statistically significant increase in the level of revertants on the test plates as compared to the control plates. The Analysis of Variance test was used
to compare the test and negative control groups. When necessary the Student's t-test was used to compare the negative and positive control groups.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Not toxic up to 5 mg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation recorded. High dose level (5 mg/plate) used 0.3 mL per plate in the preliminary toxicity test rather than 0.1 mL per
plate as usual. In the main test, a 50 mg/mL solution was used for dosing the high level (5 mg/plate) - the solution was not clear, but was judged to
be sufficiently homogeneous for dosing.
- Other confounding effects: None recorded


RANGE-FINDING/SCREENING STUDIES:
A three-fold dilution series (from 5.0 to 0.062 mg/plate) was tested with and without S-9 mix. No toxicity, measured as a colony count reduction,
was observed at any dose with or without S-9 mix.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Two statistically significant increases in revertant counts were found in TA 100 without S-9 mix. Although, the increases were only small and there was no dose-response, it was decided to perform a supplementary test to see if the increases could be confirmed. No indication of any increase was seen in the supplementary test and it was concluded that the small increases were incidental, i.e. due to incidentally low controls.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance Di-TMP was found to be non-mutagenic in the Ames test.
Executive summary:

The mutagenic potential of Di-TMP was investigated in a Ames test using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537. Triplicate cultures of all tester strains were exposed to di-TMP at concentrations up to the limit concentration of 5 mg/plate in the absence and presence of an exogenous metabolic activation system (S9 fraction). No reproducible increase in the number of revertant colonies was seen; di-TMP is therefore considered to be negative in this study. Appropriate control substances confirmed the sensitivity of this assay.