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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:
21/09/1993 to 25/10/1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to the OECD 471 test guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction Products of Diphosphorus Pentaoxide with Alcohols, C14-18 even, salted with Amines, C12-14, Tert-alkyl
EC Number:
943-540-0
Molecular formula:
Too complex
IUPAC Name:
Reaction Products of Diphosphorus Pentaoxide with Alcohols, C14-18 even, salted with Amines, C12-14, Tert-alkyl
Details on test material:
- Physical state: pale yellow viscous liquid
- Storage condition of test material: room temperature

Method

Target gene:
Histidine and tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced S9
Test concentrations with justification for top dose:
Toxicity pre-screen 50, 167, 500, 1670, 5000 ug/plate
Initial study for Salmonella 0.167, 0.5, 1.67, 5.0, 16.7, 50 ug/plate
Initial study for E.coli 16.7, 50, 167, 500, 1670, 5000 ug/plate
Confirmatory study for Salmonella 0.167, 0.5, 1.67, 5.0, 16.7, 50 ug/plate
Confirmatory study for E.coli 16.7, 50, 167, 500, 1670, 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: none given
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 minutes at 37 C
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): absence of histidine or tryptophan in agar

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: growth of background lawn of non-revertant bacteria and size and number of revertant colonies
Evaluation criteria:
A positive result is defined as a 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 spontaneous solvent control value. If the test article 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. A negative result is defined as the 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 are performed only when a 50% increase in revertant frequency, relative to the concurrent negative controls, is observed. This 50% "trigger" was selected based upon normal, spontaneous variation observed among replicate negative control cultures, as well as spontaneous fluctuation observed in this laboratory among groups of cultures treated with a variety of test articles judged to be negative in this assay.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100, WP2uvrA-
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed at 1670 ug/plate and above
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: A toxicity screening test was performed IN TA1538, TA100 and WP2uvrA-

COMPARISON WITH HISTORICAL CONTROL DATA: All control values were comparable to historical ranges

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 were negative in the Ames/Salmonella-E.coli Liquid Pre-incubation Assay under the conditions, and according to the criteria, of the test protocol.
Executive summary:

The test substance was evaluated in the Ames/Salmonella-E.coli Liquid Pre-incubation Assay to determine its ability to induce reverse mutations at selected histidine loci in five tester strains of salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100), and at the tryptophan locus in one Escherichia coli tester strain (WP2uvrA-), in the presence and absence of an exogenous metabolic activation system (S9). Toxicity of the test substance was first evaluated in a prescreen by treating duplicate culturesof strains TA1538, TA100 and WP2uvrA- with the test article at doses of 50, 167, 500, 1670 and 5000 ug/plate in the absence of S9. Results of the prescreen indicated OS#26589K was not toxic to tester strain WP2uvrA- at doses of 50, 167 and 500 ug/plate. Inhibited growth (characterised by the absence of a confluent bacterial lawn and/or the presence of pindot colonies) or complete toxicity was observed in both Salmonella strains at all doses evaluated, and in strain WP2uvrA- at dose =>1670ug/plate. In addition, the test article precipitated from solution at doses => 1670 ug/plate.

Based upon these findings, OS#26589K was evaluated in triplicate cultures in all five strains of Salmonella at doses of 0.167, 0.5, 1.67, 5, 16.7 and 50 ug/plate, and in strain WP2uvrA- at dose of 16.7, 50, 167, 500, 1670 and 5000 ug/plate, in the presence and absence of S9. Six dose levels were evaluated with and without S9 in the event of unacceptable toxicity and/or insolubility at the highest dose levels evaluated in the mutati assay. The S9 mixture included 6% (v/v) Aroclor 1254 -induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and cofactors.The test article again precipitated from solution =>1670 ug/plate. Inhited growth again was observed in all Salmonella strains at doses of 5, 16.7, and/or 50 ug/plate with and/or without S9, and in tester strain WP2uvrA- at doses of 1670 and/or 5000 ug/plate with and/or without S9. Revertant frequencies for all doses of the test substance in all tester strains with and without S9 approximated or were less than those observed in the concurrent negative control cultures.

The test substance was re-evaluated in the confirmatory assay under identical conditions, and similar results were observed. The test article again precipitated from solution at doses =>1670 ug/plate. Inhibited growth again was observed in all Salmonela strains at doses of 5, 16.7 and/or 50 ug/plate with and/or without S9, and in tester strains WP2uvrA- at doses of 1670 and/or 5000 ug/plate with and/or without S9. Revertant frequencies for all dose levels in all tester strains with and without S9 approximated or were less than control value. All positive and negative control values in both assays were within acceptable limits.

Therefore, the results for were negative in the Ames/Salmonella-E.coli Liquid Pre-incubation Assay under the conditions, and according to the criteria, of the test protocol.

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