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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:
26 June 2000 - 10 July 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed under GLP and according to standard protocol.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Cited as Directive 2000/32/EC, B.13/14
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: Halamid Pharma Grade
Chemical name: Sodium p-toluenesulfonchloramide
Description: white crystalline powder
Batch: 0299906530045
Purity: 100.2%
Test substance storage: At room temperature in the dark
Stability under storage conditions: not indicated
Expiry date: 15 June 2001 (alloctaed by Notox)
Stability in vehicle: Water: Not indicated

Method

Target gene:
histidine synthesis
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene) -
The Salmonella typhimurium strains were regularly checked to confirm their histidinerequirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
Additional strain / cell type characteristics:
other:
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The strain was regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Range-finding : 333, 1000, 3330, 5000 µg/plate.

Experiment 1:
without S-9 mix:3, 10, 33, 100, 200, 333, 1000 and 3330 µg/plate
with S-9 mix: 3, 10, 33, 100, 333, 666, 1000, 3330, 5000 µg/plate

Experiment 2:
without S-9 mix: 10, 50, 100, 150 and 200 µg/plate
with S-9 mix: 100, 200, 300, 400 and 500 µg/plate
Vehicle / solvent:
Milli-Q water
Controls
Remarks:
see below "any other information on tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: The revertant colonies (histidine independent c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present.

DETERMINATION OF CYTOTOXICITY
- Method:other: growth inhibition. Selection of an adequate range of doses was based on a dose range finding test with strain TA100 and the WP2uvrA strain, both with and without S9-mix. Eight concentrations were tested in triplicate. This dose range finding test was reported as a part of the first experiment of the mutation assay. The highest concentration of Halamid pharma grade used in the subsequent mutation assay was the level at which the test substance inhibited bacterial growth.
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a. The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
b. The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance.
c. The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
Statistics:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: All tester strains at 333 ug/plate and higher
Vehicle controls validity:
not valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
All tester strains at 333 ug/plate and higher
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: no effects
- Precipitation: no precipitation
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
To determine the toxicity of Halamid pharma grade, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. At and above 333 µg/plate reduction of the bacterial background lawn and the number of colonies was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: comparable

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
Based on the results of this study it is concluded that Halamid pharma grade is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.
Executive summary:

Halamid pharma grade was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA in two independent experiments. In the dose range finding test, Halamid pharma grade was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 -mix in the strains TA100 and WP2uvrA. Halamid pharma grade did not precipitate on the plates. In tester strain TA100, toxicity was observed at dose levels of 333 µg/plate and upwards in the absence and presence of S9 - mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 333 µg/plate and upwards in the absence of S9 -mix and at dose levels of 1000 µg/plate and upwards in the presence of S9 -mix. In the first mutation assay, Halamid pharma grade was tested up to concentrations of 200 and 666 µg/plate in the absence and presence of S9-mix respectively in the strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains. In the second mutation assay, Halamid pharma grade was tested up to concentrations of 200 and 500 µg/plate in the absence and presence of S9-mix respectively in the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Toxicity was observed in all tester strains Halamid pharma grade did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.

 

Based on the results of this study it is concluded that Halamid pharma grade is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.