Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Already evaluated by the Competent Authorities for Biocides and Existing Substance Regulations.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
The method also addressed the following guidelines:
EC Directive 2000/32 Annex V Test B14
UKEMS Guidelines
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid
Details on test material:
Lot/batch number: A668269 350
Description: Blue crystalline solid
Purity: 99.0 - 100.5%
Stability: Stable at room temperature

Method

Target gene:
See evaluation criteria.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver (Sprague-Dawley male rat) post-mitochondrial fraction (S-9 mix)
Test concentrations with justification for top dose:
1.6, 8, 40, 200, 1000 µg/plate and 50, 100, 200, 400, 800 µg/plate in mutation experiments 1 and 2, respectively.
Vehicle / solvent:
None.
Controls
Untreated negative controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Study type: Ames test.

Positive control: Details of the positive controls are in Table 1 (see 'Any other information on materials and methods').

Negative control: Yes, tests carried out with purified water in quintuplicate both with and without metabolic activation.

Administration/Exposure; Application of test substance:
Concentrations: Following a range finding study, two experiments were carried out with concentrations of 1.6, 8, 40, 200 and 1000 µg/l in experiment one and 50, 100, 200, 400 and 800 µg/l in experiment two. The tests were carried out in triplicate.

Way of application: The test article was dissolved in sterile purified water.

Pre-incubation time: Only Experiment two included a pre-incubation step for the tests with metabolic activation. The test substance (or control substance), bacteria and S-9 mix were mixed together and incubated for 1 hour at 37 °C before the addition of 2.5 ml molten agar at 46 °C. Plating of these treatments then proceeded as for normal plate-incorporation procedure.

Examinations:
Number of cells evaluated: Colonies were counted electronically using a Seescan Colony Counter and the background lawn inspected for signs of toxicity.
Evaluation criteria:
With the exception of strain TA102, these strains require biotin as well as histidine for growth. In strain TA102 the critical mutation in the histidine gene is located on a multicopy plasmid pAQ1. This strain is particularly sensitive to the activities of oxidative and cross-linking mutagens. The pKM101 plasmid derivatives (TA98, TA100 and TA102) have increased sensitivity to certain mutagens as the pKM101 codes for an error-prone DNA repair system.
Statistics:
The m-statistic was calculated to check that all the data were Poisson-distributed, and the Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

Results and discussion

Test results
Species / strain:
other: Strains TA98, TA100, TA1535, TA1537, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See additional information on results.
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Evidence of genotoxicity: There was no evidence of genotoxicity observed in either Experiment 1 or Experiment 2 in the presence or absence of metabolic activation.

Cytotoxicity: Evidence of toxicity was observed in all Experiment 1 treatments of 1000 µg/plate. Some evidence of toxicity was also observed following strain TA102 treatments of 200 µg/plate in the presence of S-9 only. In Experiment 2, toxicity was observed following all treatments (with and without metabolic activation) of 800 µg/plate. Some treatments in the presence of S-9 at lower doses also produced evidence of toxicity. The higher degree of toxicity observed with Experiment 2 treatments of S-9 was attributed to the use of a pre-incubation step, which allowed an enhanced exposure of the bacteria to the test article.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
It was concluded that copper II sulphate pentahydrate was unable to induce mutation in 5 strains of S. typhimurium, when tested at concentrations extending into the toxic range, in the absence and presence of a rat liver metabolic activation system (S-9 mix).
Executive summary:

Materials and Methods

Copper II sulphate pentahydrate was assayed for mutation in 5-histaidine requiring strains (TA98, TA100, TA1537 and TA102) of Salmonella typhimurium, both in the presence and absence of metabolic activation by Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9) in 2 separate experiments.  Following a range finding study, two experiments were carried out with concentrations of 1.6, 8, 40, 200 and 1000 µg/l in experiment one and 50, 100, 200, 400 and 800 µg/l in experiment two.  The tests were carried out in triplicate.  Both positive and negative controls were included.

The study complied with the following guidelines and was conducted in accordance with GLP:

OECD Guidelines 471
EC Directive 2000/32 Annex V Test B14
UKEMS Guidelines

 

Results and discussion

None of the dose concentrations in any of the test strains in either the absence or presence of S-9 resulted in an increase in revertant numbers that were statistically significant at the 1% level when analysed using a Dunnett’s test.  It was therefore concluded that copper II sulphate pentahydrate was unable to induce mutation in 5 strains of S. typhimurium, when tested at concentrations extending to the toxic range, in both the absence and presence of rat liver metabolic activation system.