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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: ≥ 92%- pH: ≥ 1 - ≤ 5 (aqueous preparation)
Specific details on test material used for the study:
- Name of test substance: Jodosept L- Test substance No .: 98/429-1- Batch No.: ZK 1055/175-2- Date of manufacture: June 25 - July 06, 1998- Appearance, consistency: Brown liquid- Storage: Room temperature

Method

Target gene:
his+ or trp+
Species / strain
Species / strain / cell type:
other: Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA.
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented postmitochondrial fraction (S-9) obtained from livers of rats treated with enzyme-inducing agent Aroclor 1254 .
Test concentrations with justification for top dose:
40 - 10000 µg/plate (standard plate test); 0.8 - 2500 µg/plate (preincubation test)
Vehicle / solvent:
Water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylendiamine (NOPD)
Details on test system and experimental conditions:
Standard plate test:- Salmonella typhimurium: Test tubes containing 2-mLportions of soft agar (overlay agar), which consists of 100 mL agar (0.6% agar + 0 .6% NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order: 0.1 mL test solution or vehicle, 0.1 mL fresh bacterial culture, 0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 mL phosphate buffer (in tests without metabolic activation). After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approximately 30 seconds . After incubation at 37 °C for 48-72 hours in the dark, the bacterial colonies (his+ revertants) are counted .-Escherichia coli: Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 ml agar (0.6% agar + 0 .6% NaCl) and 10 mL amino acid solution (minimal amino-acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at 45 °C, and the remaining components are added in the following order: 0.1 mL test solution or vehicle, 0.1 mL fresh bacterial culture, 0.5 mL S-9 mix (in tests with metabolic activation ) or 0.5 mL phosphate buffer (in tests without metabolic activation). After mixing, the samples are poured onto minimal agar plates within approximately 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted .Preincubation Test:0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37 °C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approximately 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.Titer determination:In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. Test tubes containing 2-mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0 .5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order: 0.1 mL vehicle (without and with test substance), 0.1 mL fresh bacterial culture (dilution : 1E-6), 0.5 mL S-9 mix. In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S-9 mix are incubated at 37 °C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured onto the agar plates within approximately 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.Mutagenicity:Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st experiment.Toxicity:Toxicity detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his or trp background growth), and a reduction in the titer is recorded for all test groups both with and without S-9 mix in all experiments.Solubility:Precipitation of the test material is recorded and indicated in the tables. As long as precipitation does not interfere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.
Rationale for test conditions:
Choice of the vehicle: Due to the good solubility of the test substance in water, water was selected as the vehicle.
Evaluation criteria:
Generally, the experiment is to be considered valid if the following criteria are met :- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.- The sterility controls revealed no indication of bacterial contamination .- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.- The titer of viable bacteria was >1E9/mL .The test chemical is considered positive in this assay if the following criteria are met:- A dose-related and reproducible increase in the number of revertant colonies, i .e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.A test substance is generally considered nonmutagenic in this test if:- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test results
Key result
Species / strain:
other: All strains tested
Remarks:
Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA.
Metabolic activation:
with and 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:
An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.A bacteriotoxic effect (reduced background growth, decrease in the number of revertants, reduction in the titer) was observed in the standard plate test from about 5,000 pg/plate onward. In the preincubation assay bacteriotoxicity was observed from about 500 µg/plate.onward.No test substance precipitation was found.Under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic agent in a bacterial reverse mutation test.

Applicant's summary and conclusion