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Additional information

In vitro Gene Mutation in Bacteria

Ames

Three read across studies are provided to address the acid component of the registered material in a weight of evidence approach. All were awarded a reliability score of 4 in accordance with the criteria set forth by Klimisch (1997). In addition, potassium sulfate tested negative in an Ames test, which was awarded a reliability of 2. This test is provided to address the potassium salt portion of the test material.

 

The mutagenic activity of the read across material sodium naphthenate was investigated in a reverse mutation test in Salmonella typhimurium using a procedure broadly in accordance with the standardised guideline OECD 471.

The test material was administered to S. typhimurium strains TA1535, TA1537, TA98 and TA100 in ethanol at concentrations of up to 3333 µg/plate. Appropriate positive and vehicle controls were used.

Under the conditions of this study, the test material was non mutagenic to Salmonella typhimurium both in the presence and absence of metabolic activation.

 

The mutagenic activity of the read across material calcium naphthenate was investigated in a reverse mutation test in Salmonella typhimurium using a procedure broadly in accordance with the standardised guideline OECD 471.

The test material was administered to S. typhimurium strains TA1535, TA1537, TA98 and TA100 in 95 % ethanol at concentrations of up to 1000 µg/plate. Appropriate positive and vehicle controls were used.

Under the conditions of this study, the test material was non mutagenic to Salmonella typhimurium both in the presence and absence of metabolic activation.

 

The mutagenic activity of the read across material calcium naphthenate was investigated in a reverse mutation test in Salmonella typhimurium and Escherichia coli using a procedure broadly in accordance with the standardised guideline OECD 471.

The test material was administered to S. typhimurium strains TA1535, TA1537, TA98 and TA100 and E. coli strains WP2 and WP2uvrA in “carrier oil” at concentrations of up to 4000 µg/plate. Appropriate positive and vehicle controls were used.

Under the conditions of this study, the test material was non mutagenic to Salmonella typhimurium and Escherichia coli both in the presence and absence of metabolic activation.

 

The mutagenic activity of the potassium salt component of the registered material was addressed in a reverse mutation test using the read across material potassium sulfate in accordance with the standardised guideline OECD 471 under GLP conditions. It was assigned a reliability score of 2 in accordance with the criteria of Klimisch (1997).

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were exposed to the test material in sterile distilled water both in the presence and absence of exogenous metabolic activation (S9-mix derived from rat liver). The bacteria were also exposed to vehicle and appropriate positive controls. The concentrations tested were 75, 200, 600, 1800, and 5000 μg/plate.

Under the conditions of this study, the test material was non mutagenic both in the presence and absence of metabolic activation.

 

In vitro Mammalian Cell Cytogenicity

Three read across studies are provided to address the acid component of the registered material in a weight of evidence approach. All were awarded a reliability score of 4 in accordance with the criteria set forth by Klimisch (1997). A chromosome aberration test on potassium sulfate, which was negative, is used to address the potassium salt portion of the test substance. The test was awarded a reliability score of 2.

 

The potential of the read across material sodium naphthenate to cause genotoxic effects was investigated in an in vitro mammalian chromosome aberration test conducted broadly in accordance with the standardised guideline OECD 473.

Chinese Hamster Ovary cells were exposed to the test material up to a maximum concentration of 250 µL in both the presence and absence of an exogenous metabolic activation system derived from the livers of rats induced with Aroclor 1254. The cells were also exposed to the appropriate solvent and positive controls.

Under the conditions of this study, the test material is non-genotoxic in the in vitro CHO Chromosomal aberration test system.

 

The potential of the read across material calcium naphthenate to cause genotoxic effects was investigated in an in vitro mammalian chromosome aberration test conducted broadly in accordance with the standardised guideline OECD 473.

Rat Liver RL4 cells were exposed to the test material up to a maximum concentration of 250 µL in the absence of an exogenous metabolic activation system. The cells were also exposed to the appropriate solvent and positive controls.

Under the conditions of this study the test material was determined to be non-mutagenic in the Rat Liver chromosomal damage test system.

 

The potential of the read across material sodium naphthenate to cause genotoxic effects was investigated in an in vitro sister chromatid exchange assay in mammalian cells conducted broadly in accordance with the standardised guideline OECD 479.

Chinese Hamster Ovary cells were exposed to the test material up to a maximum concentration of 500 µL in both the presence and absence of an exogenous metabolic activation system derived from the livers of rats induced with Aroclor 1254. The cells were also exposed to the appropriate solvent and positive controls.

Under the conditions of this study, the test material was found to be non-genotoxic in the presence of metabolic activation. Ambiguous results were obtained without metabolic activation. 

 

The mutagenic activity of the potassium salt component of the registered material was addressed in a chromosome aberration test in Chinese Hamster Ovary cells using the read across material potassium sulfate in accordance with the standardised guideline OECD 473 under GLP conditions. It was assigned a reliability score of 2 in accordance with the criteria of Klimisch (1997).

Cells were treated with the test material at concentrations of 217.5, 435, 870, and 1740 μg/mL in sterile water.

In the non-activated study, the cells were exposed to the test material for 4 hours or continuously for 20 hours up to the cell harvest. In the S9 activated study, the cells were exposed for 4 hours.

A concurrent toxicity test was conducted in both the non-activated and the S9 activated test systems and appropriate vehicle and positive controls were used.

Under the conditions of this study, the test material was judged to be non-clastogenic to Chinese Hamster Ovary cells in the presence and absence of metabolic activation.

 

In vitro Gene Mutation

The potential mutagenicity of the closely related read across material naphthenic acids potassium salts on the thymidine kinase (TK +/-) locus of the L5178Y mouse lymphoma cell line was investigated in accordance with the standardised guidelines OECD 476, EU Method B.17, EPA OPPTS 870.5300 and the Japanese METI/MHLW guidelines for testing of new chemical substances. It was assigned a reliability score of 1 in accordance with the criteria of Klimisch (1997).

Two independent experiments were performed; the maximum dose levels were limited by test material-induced toxicity. In Experiment 1, cells were treated with the test material at eight dose levels (10 to 100 µg/mL in the absence of metabolic activation, 10 to 160 µg/mL in the presence of metabolic activation), in duplicate, together with solvent and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2 % S9).

In Experiment 2, the cells were treated with the test material at eight dose levels using a 4 hour exposure group in the presence of metabolic activation (1 % S9) and a 24 hour exposure group in the absence of metabolic activation. The dose range was 2.5 to 100 µg/mL in the absence of metabolic activation and 20 to 120 µg/mL in the presence of metabolic activation.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The vehicle and positive controls were acceptable.

Under the conditions of this study, the test material is considered to be non-mutagenic in the L5178Y mouse lymphoma assay.

 

Further Information

Further to the experimental data, a Q(SAR) estimation for the read across material naphthenic acids is provided. It was assigned a reliability score of 2 in accordance with the criteria of Klimisch (1997).

The mutagenic properties of a number of proposed representative component structures of the read across material naphthenic acids were estimated. The estimations were performed using the validated CAESAR modelling tool (Istituto di Ricerche Farmacologiche Mario Negri).

Under the conditions of the CAESAR modelling tool, the test material is found to be non-mutagenic.


Justification for selection of genetic toxicity endpoint
A mouse lymphoma assy was selected as the key study as it was a K1 study and the only study that was conducted on the actual test substance. The remaining studies were used as a weight of evidence approach on the basis that multiple read across studies have been provided to address the different types of genetic toxicity.
Furthermore, this endpoint was addressed using a weight of evidence approach to structural analogues of the registered substance; studies are provided to address both the naphthenic acids component of the registered material and the potassium salt component. In this respect it is considered that the data submitted provides an adequate reflection of the test material.

Short description of key information:
IN VITRO GENE MUTATION STUDY IN BACTERIA
The read across materials sodium naphthenate, calcium naphthenate and potassium sulfate were all judged to be non-mutagenic in bacterial reverse mutation assays.

IN VITRO MAMMALIAN CELL CYTOGENICITY
The read across materials sodium naphthenate, calcium naphthenate and potassium sulfate were judged to be non-mutagenic in chromosome aberration tests.
In a sister chromatid exchange assay, the read across material sodium naphthenate was found to be non-genotoxic in the presence of metabolic activation. Ambiguous results were obtained without metabolic activation.

IN VITRO GENE MUTATION STUDY IN MAMMALIAN CELLS
The registered material was found to be non-mutagenic in a mouse lymphoma assay.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with criteria for classification as defined in Annex I, Regulation (EC) 1272/2008, the test material does not require classification for genetic toxicity based on the overall negative response noted in the available genetic toxicity studies.