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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:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
april 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and guideline conform study, read across study
Justification for type of information:
Category approach as part of CSR.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his and trp
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from aroclor induced rats and syrien hamster
Test concentrations with justification for top dose:
Range finder and first experiment 2.4, 12, 60, 300, 1500 g per plate, with and without S9
second experiment 93.75, 187.5, 375, 750, 1500 g per plate, with and without S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98 TA1538 Migrated to IUCLID6: without S9, 5 micro g per plate
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 TA1535 Migrated to IUCLID6: without S9, 2 micro g per plate
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 Migrated to IUCLID6: without S9, 50 micro g per plate
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
WP2 uvrA Migrated to IUCLID6: without S9, 2 micro g per plate
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, with S9, 5 micro g per plate
Remarks:
one strain, for standart S9 mix
Positive controls:
yes
Positive control substance:
congo red
Remarks:
for reductive S9 mix, one strain Migrated to IUCLID6: with S9, 100 micro g per plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation), second experiment with pre-incubation 30 min at 30°C

DURATION
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: in triplicate

OTHER: The S-9 used for the 'standard' plate-incorporation treatments was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 used for the 'Prival' treatments was prepared from uninduced male Golden Syrian hamsters.

range finder: tested in TA 100, in triplicate +/- S9 mix, with positive and solvent control, incorporation method

counting: Seescan Colony Counter (Seescan pic), Manual counts were performed for all treatments performed at 1500 /micro g/plate, as precipitation of the test agent on these plates prevented an accurate automated count being obtained.
Evaluation criteria:
The test article was considered to be mutagenic if:
1) the assay was valid
2) Dunnett's test gave a significant response (p < 0.01) and the data set showed a significant dose correlation
3) the positive responses described in 2) were reproducible.
Statistics:
The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis

Results and discussion

Test resultsopen allclose all
Key result
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 1500 micrgram/plate

RANGE-FINDING/SCREENING STUDIES: yes, see above under study details

COMPARISON WITH HISTORICAL CONTROL DATA:

the second experiment was done twice due to an error in the first trial (induced instead of uninduced hamster liver)

treatments of strain TA100 in Experiment 1 produced a small increase in revertant numbers (1.3 fold over the negative solvent control counts) in the presence of metabolic activation, that was however statistically significant at the 1% levelwhen the data were analysed using Dunnett's test. However, this increase was not reproduced in the second experiment (either with 'standard' or 'Prival' S-9 mix) and was therefore not attributed to mutagenic activity, but was considered to have been a chance occurrence. This study was therefore considered to have provided no clear evidence of mutagenic activity.
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

It was concluded that the test item did not induce mutation when tested in five strains of Salmonella typhimurium (TA98, TAIOO, TA1535, TA1537 and TA1538) and one strain of Escherichia coli (WP2 uvrA) under the conditions employed for this study, which included treatment up to 1500 microg/plate (a precipitating dose, approaching and/or extending into the toxic range), both in the absence and in the presence of metabolic activation systems (both 'standard' and 'reductive' S-9 mixes).