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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:
January 04, 1994 - January 14, 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study was performed only on 4 different strains instead of 5, since followed the OECD 471 of 1983, which was modified later in 1997.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Mutations of the bacterial strains used in the study can be described as follows:
Salmonella typhimirium
- TA 1537: his C 3076; rfa-; uvrB- (frame shift mutations)
- TA 98; his D 3052; rfa-; uvrB-;R-factor (frame shift mutations)
- TA 1535: his G 46; rfa-; uvrB- (base-pair substitutions)
- TA 100: his G 46; rfa-; uvrB-;R-factor (base-pair substitutions)
Regular checking of the properties of the Salmonella strains with regard to membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in the testing laboratory according to Ames test. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
Metabolic activation:
with and without
Metabolic activation system:
S9 hamster liver microsomal fraction
Test concentrations with justification for top dose:
33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 µg/plate. Concentrations covered two logarithmic decades. The choice of the top dose was based on a Range-Finding test.
Vehicle / solvent:
- Solvent: DMF
- Justification for choice of solvent: chosen to its solubility properties and its relative nontoxicity for bacteria.
Controls
Untreated negative controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see Remarks
Remarks:
Sodium azide and 4-NOPD were used for "without metabilic activation" controls, while Congo Red and 2-AA for "with metabolic activation" controls.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation).

NUMBER OF REPLICATIONS
The experiment was conducted in two independent assays. For each strain an dose level, including the controls three plates were used as a minimum.

STORAGE
Strain were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

PRECULTURES
From the thawed ampoules of the strains 0.5 ml suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre:
- 8 g Merck Nutrient Broth
- 5 g NaCl
The bacterial culture was incubated in a shaking water bath for 10 hours at 37 °C.

SELECTIVE AGAR
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri was filled with 20 ml of this nutrient medium.
The overlay agar contains per litre:
- 6.0 g Merck Agar Agar*
- 6.0 g NaCl*
- 10.5 mg L-histidine x HCl x H2O*
- 12,2 mg biotin*
* (MERCK, D-64 293 Darmstadt)
Sterilizations were performed at 121° C in an autoclave.
Statistics:
No appropriate statistical method is available since no evaluated statistical procedure can be recommended for analysis of data from the bacterial assay at this time.

Results and discussion

Test results
Species / strain:
other: TA 98, TA 100, TA 1535 and TA 1537
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:
RANGE-FINDING/SCREENING STUDIES
To evaluate the toxicity of test item, a pre-study was performed with straings TA 98 and TA 100. The plates with the test article showed normal background growth up to 5000.0 ug/plate in both the tested strains.

MAIN ASSSAYS
Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 2500.0 µg/plate (without S9 mix) and in strain TA 98 at 1000.0; 2500.0 and 5000.0 µg/plate (with and without S9 mix) in experiment I.
In experiment II toxic effects occurred at 2500.0 and 5000.0 (without S9 mix) and at 1000.0; 2500.0 and 5000.0 µg/plate (with S9 mix) in the strains TA 1535, TA 1537, and TA 98. The background growth of the bacteria (as an additional indication of toxicity) was not influenced. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

No substantial and dose-dependent increases in revertant colony numbers of any of the four tester strains were observed following treatment with test article at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in experiment I. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance in experiment II with and without S9 mix in all strains used.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Any other information on results incl. tables

Revertants/plate mean from three plates. Without S9 mix.

Concentration µg/plate TA 1535 TA 1537 TA 98  TA 100
Experiment I II I II I II I II
Neg. control 20 17 13 7 40 36 191 143
Solv. control  13 7 14 6 21 13 108 168
33.3 14 8 8 8 21 17 114 176
100.0 16 6 10 8 19 12 111 168
333.3 13 11 9 8 20 14 134 164
1000.0 10 6 10 8 11 9 101 152
2500.0 7 3 8 2 4 7 81 140
5000.0 8 2 8 1 0 4 89 164
Positive controls
Sodium azide (10 µg/plate) 850 793 734 708
4-NOPD (10 µg/plate) 78 66 453 404

Revertants/plate mean from three plates. With S9 mix.

Concentration µg/plate TA 1535 TA 1537 TA 98  TA 100
Experiment I II I II I II I II
Neg. control 15 12 22 15 35 45 156 127
Solv. control  15 15 11 17 39 53 165 140
33.3 21 15 9 18 35 56 182 144
100.0 21 17 20 18 38 50 163 128
333.3 17 17 19 11 31 43 198 106
1000.0 12 7 13 7 24 20 171 155
2500.0 10 5 9 2 10 5 226 130
5000.0 8 2 8 0 5 0 224 101
Positive controls
2-AA (2.5 µg/plate) 251 216 365 400 1530 1297
Congo Red (500 µg/plate) 263 259

Applicant's summary and conclusion

Conclusions:
The test substance did not induce gene mutations by base pair changes or frameshifts, both with and without metabolic activation.
Executive summary:

The test substance was tested for detecting its potential gene mutagenic activity using the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537. It should be noted that the study was performed only on 4 different strains of microorganisms instead of 5. This difference is due to the fact that the study was performed according to OECD 471 (1983) which was modified later in 1997.The tests were performed with and without metabolic activation. The test item was examined in two independent assays at 6 concentrations from 3.33 to 5000 µg/plate.

In the experiments performed, no relevant increase of the revertant colony numbers was observed in any Salmonella typhimurium strain tested, in the presence and in the absence of S9 mix. The test substance did not induced point mutations by base pair changes and frameshifts in the genome of the strains of Salmonella typhimurium used.