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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Neither E. coli WP2 nor S. typhimurium TA102 were tested as required in the current OECD TG 471.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted December 29, 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reference substance 003
EC Number:
611-563-2
Cas Number:
57635-48-0
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Batch No.of test material: 137304133- Expiration date of the lot/batch: 2001, JanuarySTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: romm temperature (20°C)- Stability under test conditions: Min . 24 h (0 .1% solution)- Solubility and stability of the test substance in the solvent/vehicle: soluble in waterTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: stock solutions 1. study 5000 mg/L, 2. study 50000mg/L- Preliminary purification step (if any): no- Final dilution of a dissolved solid, stock liquid or gel: in water

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: 97a
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (male wistar rats induced with Phenobarbital and ß-Naphtoflavone)
Test concentrations with justification for top dose:
cytotoxcicty was evident in TA97a without S9 in concentrations ≥ 500 µg/plate and in TA97a with S9 at 5000 µg/plateall other strains were tested up to the limit dose (5000 µg/plate)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR191 (TA97a - S9); 4-Nitro-o-phenylen-diamine (TA9 -S9); Nitrofurantoine (TA 100 - S9); 2-Aminoanthracene (TA 97a, TA 98, TA 100 + S9); Cumene Hydroperoxide (TA 102 - S9); Danthron (TA 102 + S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Exposure duration: 48 hNUMBER OF REPLICATIONS: 3DETERMINATION OF CYTOTOXICITY- Method: evaluation of the background lawn
Statistics:
Arithmetic mean values and standard deviations were calculated out of colonies per plate of three replicates .For evaluation of the results the induction rate of the mean values was calculated:revertant colonies of test substance/revertant colonies of the corresponding control = Induction rateThe test substance is to be interpretated mutagenic if there is a concentration effect relationstup and the induction rate is ≥ 2.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: 97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 ≥ 500 µg/plate, with S9 ≥ 5000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:1st study all test strains 5, 50, 500 µg/plate2nd studyTA97a - S9 16, 50, 160, 500, 1600 µg/plateTA97a + S9 16, 50, 160, 500, 1600, 5000 µg/plateTA98, TA100, TA102 ± S9 50, 160, 500, 1600, 5000 µg/plate

Applicant's summary and conclusion

Conclusions:
In this study the test substance was found to have no mutagenic effects on Salmonella typhimurium strains TA 97 a, TA 98, TA 100 and TA 102 with (+) and without (-) the metabolic activation system S9 at concentrations of 0.005 - 5.0 mg/plate.
Executive summary:
The genotoxicity of the registration substance was investigated according to the OECD Guideline 471.

The strains TA97a, TA98, TA100, and TA102 of S. Typhimurium were exposed to the test item in water at concentrations of 5 to 5000 µg/plate.

The test was performed with the pre-incubation method in all bacterial strains in both the presence and the absence of metabolic activation.

The test item was tested up to cytotoxic concentrations for S. typhimurium TA97a (500 µg/plate) or up to limit concentrations for S. typhimurium TA 98, TA100, TA102 (5000 µg/plate). Precipitation of the test substance was not detected in both the presence and the absence of metabolic activation. The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence of induced mutant colonies over background.