Oleic acid, copper salt

Genetic toxicity: in vitro

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

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

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

Test material information

Constituent 1

Reference substance name:

7758-99-8

Cas Number:

7758-99-8

IUPAC Name:

7758-99-8

Constituent 2

Reference substance name:

Cu2+ as Copper sulphate pentahydrate

IUPAC Name:

Cu2+ as Copper sulphate pentahydrate

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:

other: Salmonella typhimurium Strains TA98, TA100, TA1535, TA1537, TA102

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.

Details on test system and experimental conditions:

Study type: Ames test.

Positive control: Details of the positive controls are in Table 1.

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: Salmonella typhimurium 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

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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 ofS. typhimurium, when tested at concentrations extending to the toxic range, in both the absence and presence of rat liver metabolic activation system.