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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:
From June 24, 1988 to Sep 26, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 84-2
Deviations:
yes
Remarks:
No stability and analytical concentration of the test subsatnce was determined. However, these deviations should not significantly impact study results.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Brown particles
Details on test material:
- Name of test material (as cited in study report): MON 13900
- Physical state: Brown particles
- Analytical purity: 99.1%
- Lot/batch No.: Lot NBP 3747116 B
- Expiration date of the lot/batch: Expiration 5/92

Method

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:
S9-mix
Test concentrations with justification for top dose:
0.1, 0.3, 1, 3 and 10 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation for TA 98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
With metabolic activation for TA 98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation for TA 100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With metabolic activation for TA 100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: Sodium nitrite
Remarks:
Without metabolic activation for TA 1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation for TA 1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation for TA 1537
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation for TA 1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37±10⁰C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
Revertant colonies for plates with more than 500 revertant colonies/plate were estimated by counting revertant colonies in several fields under a stereomicroscope and multiplying the counted colonies by a factor relating the total plate area to the area of the counted fields. Revertant colonies measured in this manner are calculated to not more than three significant figures. Revertant colonies on other plates, except as noted, were counted with an Artek Model 880 automatic colony counter or counted by visual examination (<10 revertants/plate).
Evaluation criteria:
Results were considered positive for a strain/microsome combination if revertants/plate values were significantly elevated over control values (p<0.01) at three treatments and there was a statistically significant dose response (p<0.01).
Statistics:
Statistical analysis was performed on plate incorporation assay results after transforming revertant/plate values as log10 (revertants/plate). Analysis included Bartlett’s test for homogeneity of variance and comparison of treatments with controls using within-levels pooled variance and a one-sided t-test. Grubbs’ test was performed to determine if outliers were present. Statistical significance of dose response was evaluated by regression analysis for log10 transformed doses and revertants/plate.

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
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results of the statistical analyses of the plate incorporation assay results indicated that the test substance was mutagenic towards test strain TA100 in the presence and absence of S-9 mix. In combination, statistical analyses of both the initial and repeat assays for TA100 in the presence and absence of S-9 mix, indicated three treatments with revertants/plate values significantly greater than controls (p<0.01) and significant positive dose responses (p<0.01). The mutagenic response was very weak compared to other agents which are positive in this assay (2 fold over control values) and the high dose levels (3 and 10 mg/plate) were required to elicit a detectable response. The revertants/mg values calculated from the slope of the dose response curves were similar, with a range of 9.6-12.8 in the presence of S-9 and 10.9-16.1 in the absence of S-9 Mix. No mutagenic activity was indicated in these assays towards any of the other test strains used (TA98, TA1535, and TA1537). None of these other strain/microsome combinations indicated three treatments with revertants/plate values greater than controls (p<0.01) or significant dose responses (p<0.01). The repeat assay of TA98 in the absence of S-9 mix indicated one treatment with revertants/plate values significantly greater than controls (P<0.01) but with no positive dose response (P<0.01). This result was not observed in the initial assay and was not reproducible in a retest run at a narrow dose range.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

A toxicity screen was conducted using test strain TA100 with and without S-9 Mix. The test substance was not toxic at levels up to 10 mg/plate in the presence or absence of S-9 Mix. The test substance was observed to be insoluble at a level of 10 mg/plate. The maximum treatment level used in plate incorporation tests was 10 mg/plate. Some toxicity was observed in plate incorporation assays at the 10 mg/plate level. In the initial assay, toxicity was observed for TA98 and TA100 in the absence of S-9. In the repeat assays toxicity was seen for test strains TA100 in the presence and absence of S-9 mix and for TA1535 in the presence of S-9 mix.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, MON 13900 produced a very weak mutagenic response towards S. typhimurium strain TA 100 independent of the presence of a metabolic activation system. The response of greater than two fold the control was only observed at a concentration (10 mg/plate) which was toxic and at which the test substance was insoluble. The results in all other tester strain were considered to be uniformly negative.
Executive summary:

A study was conducted to determine the mutagenic potential of MON 13900 in an Ames mutagenicity assay according to EPA OPP 84-2 in compliance with GLP.

The test used S. typhimurium strains TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 mix. In the screen, no toxicity was observed for TA100 in the presence or absence of S9 at levels up to 10 mg/plate. The test substance was observed to be insoluble at 10 mg/plate and some toxicity was observed in plate incorporation assays at this dose. Mutagenic activity was observed for the test substance towards test strain TA100 in the presence and absence of S9. The combination of statistical analyses of the initial and repeat test results indicated three treatments with revertants/plate values significantly greater than controls (p < 0.01) and significant positive dose-responses (p < 0.01). Based on these results, it appears that the activity is direct-acting and not dependent on the presence of an exogenous metabolic activation system. The observed response was very weak in terms of magnitude (2-fold above controls) and compared to other agents that are positive in this assay. The potency of the response as calculated by the slope of the dose response curves gave a range of 0.003 - 0.004 revertants/nmole in the presence and absence of S9 mix for both initial and repeat assays. The repeat assay of TA98 in the absence of S9 mix indicated one treatment level with revertants/plate values significantly greater than controls (p < 0.01), with no positive dose-response. This result was not observed in the initial assay and was not reproducible in a retest run at a narrow dose range.

Under the test conditions, MON 13900 elicited a very weak mutagenic response towards S. typhimurium strain TA 100 independent of the presence of a metabolic activation system. The response of greater than 2- fold the control was only observed at a concentration which was toxic (10 mg/plate) and at which the test substance was insoluble. No reproducible mutagenic activity was observed for any of the other strains (TA 98, TA 1535 and TA 1537) in the presence or absence of metabolic activation.