Copper, .beta.-resorcylate salicylate lead complexes

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

Study period:

27 May 2010 to 20 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate coherence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Copper Lead Resorcylate Salicylate Complex

EC Number:

614-455-3

Cas Number:

68411-07-4

Molecular formula:

C7H5O4-, C7H5O3- Cu , Pb

IUPAC Name:

Copper Lead Resorcylate Salicylate Complex

Details on test material:

- Name of test material (as cited in study report): Lead-Cooper-Resorcylate-Salicylate (LC 12-15)
- Substance type: multicontituent
- Physical state: slightly pale green powder
- Composition of test material, percentage of components: copper: 11.9%, lead: 35.7%, resorcylate: 14.1%, salicylate: 35.7%
- Lot/batch No.: 09/201 (corresponding to lot N° PRESE123/
- Purity test date: 20 October 2009
- Expiration date of the lot/batch: 04 November 2011
- Storage conditions of test material: at room temperature.

Method

Target gene:

Histidine operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiments without S9 mix:
For the first experiment without S9 mix, the selected dose-levels were:
- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 1535, TA 1537, TA 100 and TA 102 strains,
- 62.5, 125, 250, 500, 1000 and 2000 µg/plate for the TA 98 strain.
The selected dose-levels for the second experiment were:
- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 100 and TA 102 strains,
- 31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 1535 and TA 1537 strains,
- 62.5, 125, 250, 500, 1000 and 2000 µg/plate for the TA 98 strain.
For the third experiment performed only with TA 102 strain, the selected dose-levels were 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate.

Experiments with S9 mix:
The selected dose-levels for both mutagenicity experiments were:
- 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 1535, TA 1537, TA 100 and TA 102 strains,
- 62.5, 125, 250, 500, 1000 and 2000 µg/plate for the TA 98 strain.

Vehicle / solvent:

Vehicle used: water for injections.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The preliminary test, all experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: thinning of the bacterial lawn, decrease in the number of revertants

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a
positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the
data obtained.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate in the TA 1537 and TA 100 strains and at dose-levels >= 1000 µg/plate in the TA 98 strain

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at dose-levels >= 500 µg/plate in the TA 1535, TA 100 and TA 98 strains and at 1000 µg/plate in the TA 1537 strain

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at dose-levels >= 250 µg/plate (second experiment) and at 500 µg/plate (third experiment)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at dose-levels >= 125 µg/plate in the TA 1537 strain, at 500 µg/plate in the TA 1535 and TA 100 strains, and at 2000 µg/plate in the TA 98 strain

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at dose-levels >= 250 µg/plate in the TA 1537 and TA 100 strains, at 500 µg/plate in the TA 1535 strain, and at 2000 µg/plate in the TA 98 strain

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at dose-levels >= 250 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
During the solubility assay, the test item was found not soluble in the vehicles usually used for this type of assay. Consequently, a suspension was
selected for the treatment. An homogenous suspension (to the naked eye) was obtained in water for injections at the concentration of 50 mg/mL.
Using a treatment volume of 100 µL/plate, the highest dose-level tested in the preliminary test was 5000 µg/plate, which is the highest dose-level
recommended in the international guidelines. Successive dilutions of the stock preparation were also performed in water for injections and used with a treatment volume of 100 µL/plate. Therefore the dose-levels tested in the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

For the preliminary test, the test item was first suspended in water for injections at 50 mg/mL, and then successive dilutions of this stock preparation were performed. Using a treatment volume of 100 µL/plate, the dose-levels tested in the preliminary test were 10, 100, 500, 1000, 2500 and 5000 µg/plate; the latter being the highest dose-level recommended in the international guidelines.

A moderate to strong precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 100 µg/plate.

Regarding the toxicity of the test item towards the bacteria strain, a moderate thinning of the bacterial lawn was noted at dose-levels >= 500 µg/plate and a strong decrease in the number of revertants at dose-levels >= 2500 µg/plate in the TA 98 strain either with or without S9 mix.
In the TA 100 and TA 102 strains, a strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn was observed at
dose-levels >= 500 µg/plate either with or without S9 mix.


COMPARISON WITH HISTORICAL CONTROL DATA:
- the number of revertants in the vehicle controls was consistent with the historical data of the testing facility;
- the number of revertants in the positive controls was consistent with the historical data of the testing facility.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: incorporation plate method

Any other information on results incl. tables

The sterility of the test item (stock preparation) was checked and found satisfactory.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item Lead-Copper-Resorcylate-Salicylate (LC12-15) did not show any mutagenic activity in the bacterial reverse mutation test with
Salmonella typhimurium, either in the presence or in the absence of metabolic activation.

Executive summary:

The objective of this study was to evaluate the potential of the test item Lead‑Copper‑Resorcylate-Salicylate (LC12-15) to induce reverse mutation in Salmonella typhimurium.

The study was performed according to the international guidelines (OECD guideline No. 471 and Commission Directive No. 200/32/EC, B13/14) and in compliance with the principles of Good Laboratory Practice.

 

Methods

 

A preliminary toxicity test was performed to define the dose-levels of Lead-Copper-Resorcylate-Salicylate (LC12-15) to be used for the mutagenicity study. The test item was then tested in three independent experiments, with and/or without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes,).

 

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The dose-levels of the positive controls were as follows:

 

without S9 mix

.            1 µg/plate of sodium azide (NaN₃): TA 1535 and TA 100 strains,

.            50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

.            0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

.            0.5 µg/plate of Mitomycin C (MMC): TA 102 strain.

 

with S9 mix

.            2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537 and TA 98 strains,

.            5 µg/plate of Benzo(a)pyrene (BAP): TA 100 strain,

.            10 µg/plate of 2-Anthramine (2AM): TA 102 strain.

 

Results

 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

 

The test item Lead-Copper-Resorcylate-Salicylate (LC12-15) was suspended in water for injection.

 

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix

For the first experiment without S9 mix, the selected dose-levels were:

.            15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 1535, TA 1537, TA 100 and TA 102 strains,

.            62.5, 125, 250, 500, 1000 and 2000 µg/plate for the TA 98 strain.

A moderate precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 125 µg/plate.

A moderate to strong toxicity was noted at dose-levels >= 500 µg/plate in the TA 1537, TA 100 and TA 102 strains and at dose-levels >= 1000 µg/plate in the TA 98 strain.

 

The selected dose-levels for the second experiment were:

.            15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 100 and TA 102 strains,

.            31.3, 62.5, 125, 250, 500 and 1000 µg/plate for the TA 1535 and TA 1537 strains,

.            62.5, 125, 250, 500, 1000 and 2000 µg/plate for the TA 98 strain.

A moderate precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 62.5 µg/plate in the TA 100 strain, at dose-levels >= 125 µg/plate in the TA 1535, TA 1537 and TA 102 strains and at dose-levels >= 250 µg/plate in the TA 98 strain.

For the third experiment performed only with TA 102 strain, the selected dose-levels were 15.6, 31.3, 62.5, 125, 250 and 500 µg/plate. A moderate precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 125 µg/plate. A moderate to strong toxicity was noted at dose-levels >= 500 µg/plate.

 

Slight increases in the number of revertants (up to 2.2-fold the vehicle control value) were noted in the TA 102 strain in the first experiment. These increases exceeded the threshold of 2-fold the vehicle control value at the dose-level of 62.5 µg/plate. But, since these increases were not observed in the second and third experiments performed under the same experimental conditions, they were considered to not be biologically significant.

The test item did not induce any other noteworthy increase in the number of revertants in the other strains tested in the absence of metabolic activation.

 

Experiments with S9 mix

The selected dose-levels for both mutagenicity experiments were:

.            15.6, 31.3, 62.5, 125, 250 and 500 µg/plate for the TA 1535, TA 1537, TA 100 and TA 102 strains,

.            62.5, 125, 250, 500, 1000 and 2000 µg/plate for the TA 98 strain.

 

In the first experiment performed using the direct plate incorporation method, a moderate precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 125 µg/plate. Moreover, a moderate to strong toxicity was noted at dose-levels >= 125 µg/plate in the TA 1537 strain, at dose-levels >= 500 µg/plate in the TA 1535, TA 100 and TA 102 strains, and at 2000 µg/plate in the TA 98 strain.

In the second experiment performed using the preincubation method, a moderate precipitate was observed in the Petri plates when scoring the revertants at dose-levels >= 62.5 µg/plate in the TA 98 and TA 100 strains and at dose-levels >= 125 µg/plate in the TA 1535, TA 1537 and

TA 102 strains. Moreover, a moderate to strong toxicity was noted at dose-levels >= 250 µg/plate in the TA 1537, TA 100 and TA 102 strains, at dose-levels >= 500 µg/plate in the TA 1535 strain, and at 2000 µg/plate in the TA 98 strain.

 

In the presence of metabolic activation, the test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Conclusion

Under the experimental conditions of the study, the test item Lead-Copper-Resorcylate-Salicylate (LC12-15) did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium,either in the presence or in the absence of metabolic activation.