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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:
1989-01-31 to 1989-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
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): Genaminox LA
- Composition of test material, percentage of components: 30% lauryldimethylaminoxide; 70% water
- Lot/batch No.: 88/1307
-pH value in water: 6-8
- Storage condition of test material: Dark at 22 degrees Centigrade

Method

Target gene:
Histidine for Salmonella typhimurium
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 100, TA 1535, TA 1537, TA 1538 and TA 98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: All five strains are deficient in complete structure of their lipopolysaccharide layer and in DNA excision repair system
Metabolic activation:
with and without
Metabolic activation system:
S-9 rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
Zero to 10,000 microgram/plate (see Tables 1-10, attached)
Vehicle / solvent:
Compound was dissolved in 100 uL DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix

Migrated to IUCLID6: TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene with TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation

Migrated to IUCLID6: TA 100 and TA 1535
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation

Migrated to IUCLID6: TA 1537
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation

Migrated to IUCLID6: TA 98 and TA 1538
Details on test system and experimental conditions:
Preparation and storage of liver homogenate fraction (S-9)

Male Sprague Dawley rats (200-300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation was performed at 0 to 4 degrees Centigrade using cold sterile solution and glassware. The livers from at least 5-6 animals were removed and pooled before washing in 150 mM KCl (approximately 1 mL/g wet livers). The washed livers were cut into small pieces and homogenized in three volumes of KCl. The homogenate was then centrifuged at 9000 g for 10 minutes. The supernatent (the S-9 fraction) was divided into small portions, frozen rapidly and stored at -80 degrees Centigrade for not longer than three months.

Preparation of S-9 Mix

Sufficient S-9 fraction was thawed immediately before each test at room temperature. One volume of S-9 fraction was mixed with 9 volumes of the S-9 cofactor solution and kept on ice until required. That preparation was termed the S-9 mix and the composition was defined as follows: 8mM magnesium chloride; 33 mM potassium chloride; 5 mM glucose-6-phosphate; 4 mM NADP+; 100 mM phosphate buffer pH 7.4.

Bacteria

Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2/L) at 37 degrees Centigrade. A presence of a suitable amount of bacteria in the cell suspension was checked by nephelometry. For inoculation, stock cultures stored at -80 degrees Centigrade were used and the various bacterial strains were periodically identified.

Toxicity experiments and dose range finding

The first experiment was performed with all tester strains using three plates per dose to obtain information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occurring colonies was used as an indicator for toxicity together with visible thinning of the bacterial lawn. Thinning of the bacterial lawn was controlled microscopically.

In combination with the second experiment, toxicity testing was performed as follows: 0.1 mL of the relevant test compound dilution was thoroughly mixed with 0.1 mL of 10E-6 dilution the overnight culture of TA 100 and plated onto histidine and biotin rich top agar (3 plates per dose).

Mutagenicity test

Top agar was prepared for the Salmonella strains by mixing 100 mL agar (0.6% agar, 0.5 % sodium chloride) with 10 mL of a 0.5 mM histidine-biotin solution. The following ingredients were added (in order) to 2 mL of molten top agar at 45 degrees Centigrade: 0.1 mL of an overnight nutrient broth culture of the bacterial tester strain; 0.1 mL test compound solution; 0.5 mL S-9 mix (if required) or buffer. After mixing, the liquid was poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose).
Evaluation criteria:
The solvent control was compared with the number of colonies per plate in the presence of the test compound. Results were then presented as a ratio of these values (equal to surviving fraction). For the mutagenicity test, colonies (his+ revertants) were counted after incubation for 48 to 72 hours at 37 degrees Centigrade in the dark.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
other: S. typhimurium TA 100, TA 1535, TA 1537, TA 1538 and TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 100, TA 1535, TA 1537, TA 1538 and TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Referring to Tables 1-5 (attached), the test compound proved to be toxic to the bacterial strains at a dose of 2,500 microgram per plate with metabolic activation and very toxic to the bacterial strains at a dose of 20 microgram/plate without metabolic activation. For mutagenicity testing, 2,500 microgram/plate was chosen as the highest dose in the second experiment.

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the the absence or presence of S-9 mix. No dose dependent effect is shown by the results presented in Tables 6-10 (attached).
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 test substance is not mutagenic in the presence or absence of an exogenous metabolizing system