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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:
2003-10-07 to 2003-10-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restriction

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 19, 2000 with Annex 4D
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2003-07-01
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): BIO 377
- Chemical name: 1,3-Benzenediol, 4-(1-phenylethyl)-
- Physical state: viscous, clear
- Storage condition of test material: cool, dry and dark
No further information on the test material was stated.

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
I. Experiment (AM015031): 1.5, 5, 15, 50, 150, 500 µg/L (with and without S9-mix)
II. Experiment (AM015032): 1.5, 5, 15, 50, 150, 300, 500 µg/L (without S9-mix)
II. Experiment (AM015032): 1.5, 5, 15, 50, 150, 500, 1000 µg/l (with S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [DMSO]; the solutions were freshly prepared just prior to use.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
with and without S9-mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, 50 µl/plate, with and without S9-mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene, 0.8 µg/plate for TA98 and TA100, 0.9 µg/plate for TA102 and TA 1535, 1.7 µg/plate for TA1537
Remarks:
with S9-mix
Untreated negative controls:
yes
Remarks:
with and without S9-mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, 50 µl/plate, with and without S9-mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9-mix

Migrated to IUCLID6: 50 µg/plate for TA1537, dissolved in distilled water
Untreated negative controls:
yes
Remarks:
with and without S9-mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, 50 µl/plate, with and without S9-mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix

Migrated to IUCLID6: 0.15 µg/plate for TA 102 (Exp. I) and 0.25 µg/plate (Exp. II), dissolved in distilled water
Untreated negative controls:
yes
Remarks:
with and without S9-mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, 50 µl/plate, with and without S9-mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9-mix

Migrated to IUCLID6: 2.5 µg/plate for TA98, dissolved in distilled water
Untreated negative controls:
yes
Remarks:
with and without S9-mix
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, 50 µl/plate, with and without S9-mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9-mix

Migrated to IUCLID6: 0.7 µg/plate for TA100 and TA1535, dissolved in distilled water
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (standard plate incorporation assay)

DURATION
- Exposure duration: 48 to 72 hr in the dark

NUMBER OF REPLICATIONS: 3

EVALUATION
- The frequency of revertant colonies was assessed by counting.

DETERMINATION OF CYTOTOXICITY
- Method: background lawn

OTHER EXAMINATIONS:
- The plates were examined for existence of a normal background lawn and/or precipitates and microscopically for microcolony growth.

OTHER:
- Cell titre was determined using 0.1 mL aliquots of the 10E-06 dilution, spread over the surface of complete medium plates and incubated overnight at 37 °C,
- The experiment was repeated in full after an interval of at least 3 days.
Evaluation criteria:
According to OECD 471, adopted 1997.
Statistics:
Chi square-Test was used for the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dose level.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9-mix BIO 377 was bacteriotoxic towards all tester strains at 500 µg/L plate. In the presence of S9-mix BIO 377 was bacteriotoxic towards the strains TA1535, TA1537, TA100, and TA102 at 500 µg/L plate and towards TA98 at 1000 µg/L plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS:
- The test compound failed to induce significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system.
- The estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a Chi square-test did not reveal a significant effect at any one of the test points.
- The negative result from the incorporation test was confirmed by a second independent experiment (plate incorporation assay).

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance on the plates was not observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of spontaneous revertants observed using each of the five strains is close to those previously established in the laboratory and is within the range obtained by AMEs et.al. (1975) as well as by Mortelmans and Zeiger (2000). Similarly, the results with the positive control substances confirm the known reversion properties and specificity of the test strains as well as the full activity of the metabolizing system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the absence of S9-mix BIO 377 was bacteriotoxic towards all tester strains at 500 µg/L plate.
- In the presence of S9-mix BIO 377 was bacteriotoxic towards the strains TA1535, TA1537, TA100, and TA102 at 500 µg/L plate and towards TA98 at 1000 µg/L plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The results indicate that the test substance (BIO 377 ) under the experimental conditions described, is not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA 102 in the presence and absence of a metabolising system.