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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: proposed revised testing guidelines developed by OECD
Version / remarks:
Gatehouse et al., 1994
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Independent confirmatory assay should be performed under identical rather than alternate treatment conditions as performed in the present study.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: White powder
- Storage: refrigerated at -4°C

Method

Target gene:
Salmonella strains: Histidine
Escherichia coli: Tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (liver homogenate from Aroclor 1254-induced male rats), 6%
Test concentrations with justification for top dose:
Toxicity pre-test (TA1537, TA100, WP2 uvrA, -S9 mix): 50, 167, 500, 1670 and 5000 µg/ plate;
Liquid pre-incubation assay (+/- S9 mix): 1.67, 5.00, 16.7, 50.0, 100 and 167 µg/ plate;
Plate incorporation assay (-S9 mix): 0.167, 0.5, 1.67, 5.00, 16.7 and 33.3 µg/ plate;
Plate incorporation assay (+S9 mix): 0.5, 1.67, 5.00, 16.7, 33.3 and 50 µg/ plate;
Re-test of TA1535 and TA100, liquid pre-incubation assay (- S9 mix): 0.167, 0.5, 1.67, 5.00, 16.7 and 33.3 µg/ plate;
Results of the following tests are described, but data are not included in the report:
Re-test (all strains) plate incorporation assay (+ S9 mix): 1.67, 5.00, 16.7, 50, 167 and 333 µg/ plate;
Re-test (all strains) plate incorporation assay (- S9 mix): 1.67, 5.00, 16.7, 50, 100, and 167 µg/ plate.
Vehicle / solvent:
- Vehicle solvent used: DMSO
- Dilutions were prepared on the day of the test and used immediately after preparation
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
other: 2-Nitrofluorene
Remarks:
In absence of metabolic activation
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Aminofluorene
Remarks:
In presence of metabolic activation
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Anthramine
Remarks:
In presence of metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: Two independent main studies were conducted, one following plate incorporation protocol and one with liquid pre-incubation.

DETERMINATION OF CYTOTOXICITY: Inhibition of growth was characterized by the absence of a confluent bacterial lawn and/or presence of pindot colonies.
Rationale for test conditions:
The toxicity of the test article was first evaluated in a prescreen, using both liquid pre-incubation and plate incorporation treatment conditions, by evaluating the growth of the background lawn and/or frequency of spontaneous revertants. Each dose was tested in duplicate.
Evaluation criteria:
- Positive result: statistically significant, dose-dependent increase in the number of histidine- or tryptophan-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the solvent control value.
- If the test substance does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal.
- Negative result: absence of a statistically significant or dose-dependent increase in the number of histidine or tryptophan-independent revertants.
Statistics:
Statistical analyses were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit. Statistical analyses were performed only when a 50% increase in revertant frequency, relative to the concurrent negative controls, is observed.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 16.7 µg/ plate and above (without S9-mix), at 100 µg/ plate (with S9-mix) in liquid pre-incubation assay, no cytotoxicity in plate incorporation assay.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 16.7 µg/ plate and above (without S9-mix), at 100 µg/ plate (with S9-mix) in liquid pre-incubation assay, no cytotoxicity in plate incorporation assay.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 50 µg/ plate and above (without S9-mix), at 167 µg/ plate (with S9-mix) in liquid pre-incubation assay, no cytotoxicity in plate incorporation assay.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 16.7 µg/ plate and above (without S9-mix), at 100 µg/ plate (with S9-mix) in liquid pre-incubation assay, no cytotoxicity in plate incorporation assay.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 50 µg/ plate and above (without S9-mix), at 167 µg/ plate (with S9-mix) in liquid pre-incubation assay, no cytotoxicity in plate incorporation assay.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 50 µg/ plate and above (without S9-mix), at 167 µg/ plate (with S9-mix) in liquid pre-incubation assay, no cytotoxicity in plate incorporation assay.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Revertant frequencies for all doses of N-0923 in all tester strains with and without metabolic activation under liquid pre-incubation conditions approximated or were less than those observed in the concurrent solvent control cultures. Similar results were observed under plate incorporation conditions in tester strains TA1535, TA1537, TA98, TA102 and WP2 uvrA with and without S9-mix. Statistically significant increases in revertant frequencies (appr. 1.4 to 2.1-fold increase compared to controls) were observed in TA100 at doses of 0.5 to 16.7 µg/ plate with S9-mix, and at doses of 0.5 and 1.67 µg/ plate without S9-mix under plate incorporation conditions. These increases were not linear, and all but one of the observed revertant frequencies were within historical negative control values.
In the re-tests (data described, but not included in the report), revertant frequencies for all doses of N-0923 in both tester strains without S9 under liquid pre-incubation conditions approximated or were below control values. Similar results were observed under plate incorporation conditions in tester strains TA1537, TA98, TA102 and WP2 uvrA with and without S9-mix. A statistically significant increase, to approximately 2.5-fold control values), was observed for TA1535 at 1.67 µg/ plate without S9-mix. This increase was not linear, and the observed revertant frequency was within historical negative control values.
Based on fact that observed increases were very slight, on the lack of linearity of the effects and based on absence of reproducibility the increases observed in TA100 in the first assay, and in TA1535 in the retest are considered not related to genotoxic effect but rather the result of random fluctuation of the spontaneous revertant frequencies.
All positive and solvent control values in all assays were within acceptable ranges.

Any other information on results incl. tables

Results of the prescreen indicated that N-0923 was not toxic to TA1537 at a dose of 50 µg/ plate under plate incorporation conditions (no metabolic activation). Inhibited growth or complete toxicity was observed in TA1537, TA100 and WP2 uvrA at all other doses. No precipitation was observed.

Applicant's summary and conclusion

Conclusions:
In an AMES test, conducted equivalent to OECD guideline 471, N-0923 was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was conducted with N-0923 equivalent to OECD guideline 471. Two independent main studies were conducted, one following plate incorporation protocol and one with liquid pre-incubation with test strains TA1535, TA 1537, TA98, TA100, TA102 and WP2 uvrA. All positive and solvent control values in all assays were within acceptable ranges, it was thus concluded that the test system functioned properly. Revertant frequencies for all doses of N-0923 in all tester strains with and without metabolic activation under liquid pre-incubation conditions approximated or were less than those observed in the concurrent solvent control cultures. Similar results were observed under plate incorporation conditions in tester strains TA1535, TA1537, TA98, TA102 and WP2 uvrA with and without S9-mix. Statistically significant increases in revertant frequencies (appr. 1.4 to 2.1-fold increase compared to controls) were observed in TA100 at doses of 0.5 to 16.7 µg/ plate with S9-mix, and at doses of 0.5 and 1.67 µg/ plate without S9-mix under plate incorporation conditions. These increases were not linear, and all but one of the observed revertant frequencies were within historical negative control values.

In the re-tests, revertant frequencies for all doses of N-0923 in both tester strains without S9 under liquid pre-incubation conditions approximated or were below control values. Similar results were observed under plate incorporation conditions in tester strains TA1537, TA98, TA102 and WP2 uvrA with and without S9-mix. A statistically significant increase, to approximately 2.5-fold control values), was observed for TA1535 at 1.67 µg/ plate without S9-mix. This increase was not linear, and the observed revertant frequency was within historical negative control values. Based on fact that observed increases were very slight, on the lack of linearity of the effects and based on absence of reproducibility the increases observed in TA100 in the first assay, and in TA1535 in the retest are considered not related to genotoxic effect but rather the result of random fluctuation of the spontaneous revertant frequencies. Based on these results it is concluded that N-0923 was not mutagenic with or without metabolic activation.