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Genetic toxicity in vitro

Description of key information

No experimental in vitro genetic toxicity studies were available for Naphthenic acids, however Weight of Evidence was available from ‘Sodium naphthenates’:

- Salmonella bacterial mutagenicity: negative up to >333 μg/L with and without metabolic activation (NTP, 1993; HPVIS, 2012).

- Chromosomal aberration: negative in CHO cells at 54, 116 & 250 μg/mL without metabolic activation and 25, 54, 116 & 250 μg/mL with metabolic activation (NTP, 1993; HPVIS, 2012).

- Sister Chromatid Exchange: weakly positive to positive when tested at concentrations of 17, 59, 167, 500 ug/mL (Trial 1; without metabolic activation) and 100, 150, 200, 250 µg/mL (Trial 2; without metabolic activation) and negative at 17, 59, 167, 500 µg/mL with metabolic activation (NTP, 1993; HPVIS, 2012). Although a positive result is obtained in 2 separate runs without metabolic activation, the validity of these results is questionable since the occurrence of cytotoxicity is not well documented.

 Further Weight of Evidence is available from ‘Calcium naphthenates’:

- E-coli and Salmonella bacterial reverse mutagenicity: negative in WP2 uvr A and Salmonella TA 1535, TA 1537, TA 98 and TA 100 strains when tested at 31.25 - 4000 μg/plate with and without metabolic activation (Shell Research Ltd, 1983).

- Saccharomyces cerevisiae: non-mutagenic when tested at 10 -5000 μg/plate with and without metabolic activation (Shell Research Ltd, 1983).

- Rat Liver chromosomal damage: non-mutagenic at 62.5-250 μg/mL without metabolic activation (Shell Research Ltd, 1983).

- In vitro testing in L5178Y T K +/-mouse lymphoma cells both with and without metabolic activation at 0.0005 to 10000 µg/mL showed a positive effect in the absence of metabolic activation (Seifried et al, 2006), however when studying the raw data and the evaluation criteria the applicant can not support this conclusion.

 Additional Weight of Evidence is available from QSAR prediction on the various molecules (C6 -C30 chain lengths):

- VEGA QSAR model which is an extension of the original CAESAR model (Ferrari & Gini, 2010; Benigni et al;, 2008): consistently non-mutagenic.

- Toxtree: Benigni-Bossa rulebase for mutagenicity (Benigni et al., 2008; Benigni et al., 2007): consistently non-mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1993
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
All studies have been performed in compliance with the standard procedures of the US National Toxicology Program. However, no full reports are available form the NTP database and verly limited or no data is available about cytotoxicity by which the interpretation of the results on an individual basis is very difficult.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
induced male Syrian hamster liver S9 and induced male Sprague Dawley rat liver S9
Test concentrations with justification for top dose:
1 - 3333 ug/L depending upon strain
Vehicle / solvent:
100% Ethanol
Details on test system and experimental conditions:
Info from NTP website:

Several strains of the S. typhimurium bacterium may be used for testing. Each is genetically different, so using several strains in a test increases the opportunity of detecting a mutagenic chemical. The most frequently used strains are TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538. In addition to the Salmonella tester strains, the NTP has recently begun to routinely employ Escherichia coli strain WP2 uvrA pKM101 as a bacterial tester strain in the Ames test. This E. coli strain is similar in mutagen detection to S. typhimurium strain TA102. All the bacterial strains used in the Ames test carry a defective (mutant) gene that prevents them from synthesizing the essential amino acid histidine from the ingredients in standard bacterial culture medium. Therefore, these "tester" strains can only survive and grow on medium that contains excess histidine. However, in the presence of a mutagenic chemical, the defective histidine gene may be mutated back to the functional state, allowing the bacterium to grow on standard medium that does not contain supplemental histidine. These mutations, which lead to a regaining of normal activity or function, are called "back" or "reverse" mutations and the process is referred to as "reversion." The mutant colonies, which can make histidine, are called "revertants." (There are other mutagenicity assays using other cell-types that measure "forward" mutations, that is, mutations that alter a functional gene in a way that causes a loss, rather than a gain, of function.)

In the standard protocol (preincubation) for conducting the Ames assay, a test tube containing a suspension of one strain of Salmonella typhimurium (or E. coli) plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical. Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are prepared; these contain the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen*. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow. These colonies are comprised of bacteria that have undergone reverse mutation to restore function of the histidine-manufacturing gene. The number of colonies is usually counted after 2 days.

Several modifications of the Ames test protocol have been used over the years in special circumstances. These include standard plate incorporation (no preincubation step prior to plating onto Petri dishes), FMN reduction (use of flavin mononucleotide for reduction of test articles such as azo dyes), plate test with volatile liquids (exposure of bacteria in a sealed Petri dish), cecal reduction (use of rat cecal bacteria to provide reduction of azo compounds), and plate tests conducted within a sealed dessicator (gas chamber) for exposure to gaseous substances. The specific test protocol that was used in an Ames test is noted in the description of the assay data.

Spontaneous mutations (those that occur by chance, not by chemical treatment) will appear as colonies on the control petri dishes. If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain of bacteria. The positive control plates are also counted, and the number of mutant colonies appearing on them must be significantly increased over the spontaneous control number for the test to be considered valid. Failure of the positive control chemical to induce mutation is reason to discard the experiment.

Several doses (usually at least 5) of each test chemical and multiple strains of bacteria are used in each experiment. In addition, cultures are set up with and without added liver S9 enzymes at varying concentrations. Therefore, a variety of culture conditions are employed to maximize the opportunity to detect a mutagenic chemical. In analyzing the data, the pattern and the strength of the mutant response are taken into account in determining the mutagenicity of a chemical. All observed responses are verified in repeat tests. If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be nonmutagenic in the Ames test.

*Positive control chemicals used in NTP Ames tests:

For strains tested in the absence of S9
TA98, 2-nitrofluorene or alternatively, TA98 and TA1538, 4-nitro-o-phenylenediamine
TA100 and TA1535, sodium azide
TA97 and TA1537, 9-aminoacridine
TA102, mitomycin C
TA104, methyl methanesulfonate
E.coli WP2 uvrA pKM101, methyl methanesulfonate

For strains tested with S9
All strains, 2-aminoanthracene (or occasionally, sterigmatocystin)


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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

See original document with results

Conclusions:
Sodium Naphthenate is non-mutagenic in the Salmonella bacterial Mutagenicity test system.
Executive summary:

Sodium Naphthenate was non-mutagenic in the Salmonella bacterial Mutagenicity test system strains TA100, TA1535, TA97, TA98 when tested up to >333 µg/L with and without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
All studies have been performed in compliance with the standard procedures of the US National Toxicology Program. However, no full reports are available form the NTP database and verly limited or no data is available about cytotoxicity by which the interpretation of the results on an individual basis is very difficult.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO-W-B1
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Induced Rat Liver S9
Test concentrations with justification for top dose:
54, 116 & 250 μg/mL without metabolic activation and at 25, 54, 116 & 250 μg/mL with metabolic activation
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9 activation
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 activation
Details on test system and experimental conditions:
Because many birth defects and most cancers are associated with abnormal chromosome complements, it is important to identify chemicals that can induce chromosome damage. To assess induction of CA, cells were harvested in their first mitotic division after the initiation of chemical exposure. Therefore, in the CA test without S9, cells were incubated for 8-12 hours with the test chemical in McCoy's 5A medium supplemented with fetal calf serum, L-glutamine, and antibiotics; Colcemid was added and incubation continued for 2 hours. The incubation time and the dose levels selected were determined from the information on cell cycling and toxicity obtained from the SCE test; if cell cycle delay was anticipated in the CA test, the incubation period was extended to permit accumulation of sufficient cells in first metaphase for analysis. The cells were harvested by mitotic shake-off, fixed, and stained with Giemsa. For the CA test with S9, cells were treated with the test chemical and S9 for 2 hrs, after which the treatment medium was removed and the cells incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hrs. Cells were harvested in the same manner as for the treatment without S9.

Evaluation criteria:
Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 +/- 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. One hundred or two hundred first-division metaphase cells were scored at each dose level. The classes of aberrations that were recorded included "simple" (breaks and terminal deletions), "complex" (rearrangements and translocations), and "other" (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

Statistics:
Chromosomal aberration data are presented as the percentage of cells with aberrations. To arrive at a statistical call for a trial, analyses were conducted to assess the presence of a dose-response (trend test) and the significance of the individual dose points compared to the vehicle control (Galloway et al., 1987). For a single trial, a statistically significant (P<0.05) difference for one dose point and a significant trend (P<0.015) was considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A strong trend (P < 0.003) with a single significant dose level was designated weak positive *, to indicate a high level of induced aberrations. A strongly positive trend (P < 0.003), in the absence of a statistically-significant increase at any one dose point, led to an equivocal call. Ultimately, the trial calls were based on a consideration of the statistical analyses as well as the biological information available to the reviewers. Trials that gave a weak positive or positive result were repeated. The overall result for the CA assay was based on an evaluation of the responses in all trials within an activation condition.


Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

See original document with results

Conclusions:
Sodium naphtenate was non-genotoxic in the in vitro CHO Chromosmal aberration test system.
Executive summary:

Sodium napthhenate was non-genotoxic in the in vitro CHO Chromosmal aberration test system tested at concentrations of 54, 116 & 250 μg/mL without metabolic activation and at 25, 54, 116 & 250 μg/mL with metabolic activation.

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
All studies have been performed in compliance with the standard procedures of the US National Toxicology Program. However, no full reports are available form the NTP database and verly limited or no data is available about cytotoxicity by which the interpretation of the results on an individual basis is very difficult.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay in mammalian cells
Target gene:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CHO-W-B1
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Induced Rat Liver S9
Test concentrations with justification for top dose:
Without activation: 17, 59, 167, 500 ug/ml (Trial 1); 100, 150, 200, 250 ug/ml (Trial 2); With activation: 17, 59, 167, 500 ug/ml.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9 activation
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 activation
Details on test system and experimental conditions:
Sister chromatid exchanges are a measure of DNA damage and increased levels of DNA damage are associated with mutation induction and cancer. Assaying for SCE requires examining cells that have entered their second mitotic division after the initiation of chemical exposure. Therefore, in the SCE test without S9, CHO cells were incubated with the test chemical for 26 hours in McCoy's 5A medium supplemented with fetal calf serum, L-glutamine, and antibiotics. 5-Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium with test chemical was removed and replaced with fresh medium plus BrdU and Colcemid, without test chemical. Incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with the test chemical, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum and BrdU and no test chemical. Incubation proceeded for an additional 26 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9.
Evaluation criteria:
All slides were scored blind and slides from a single test were read by the same person. Fifty second-division metaphase cells were scored to determine the frequency of SCE/cell for each dose level. If significant chemical-induced cell cycle delay was seen in treated cultures, the incubation time was lengthened to ensure the accumulation of a sufficient number of scorable (second-division metaphase) cells.
Statistics:
Statistical analyses were conducted to assess the presence of a dose-response (trend test) and the significance of the individual dose points compared to the vehicle control (Galloway et al., 1987). An SCE frequency 20% above the concurrent solvent control value was chosen as a statistically conservative positive response (Galloway et al., 1985). The probability of this level of difference occurring by chance at one dose point is less than 0.01; the probability for such a chance occurrence at two dose points is less than 0.001. An increase of 20%, or greater, at any single dose, was considered weak evidence of activity (weak positive); increases at two or more doses resulted in a determination that the trial was positive. A trend P-value of less than 0.005, in the absence of any responses reaching 20% above background, led to a call of equivocal for the trial. Positive and weak positive trials were repeated. The overall assay result was based on an evaluation of the responses in all trials within an activation condition.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: Results do not show cytotoxicity as such, however, indirect indications of cytotoxicity can be derived from the results.
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

See original document with results

Conclusions:
ambiguous without metabolic activation

Weakly positive (trial 1- without metabolic activation); Positive (trial 2 - without metabolic activation); Negative (with metabolic activation).
Executive summary:

Sodium napthhenate was weakly positive to positive (trial 1 and 2- without metabolic activation) and negative (with metabolic activation) in the in vitro SCE Sister Chromatid Exchange test system tested at concentrations of 17, 59, 167, 500 ug/mL (Trial 1; without metabolic activation) and 100, 150, 200, 250 ug/mL (Trial 2; without metabolic activation) and 17, 59, 167, 500 ug/mL with metabolic activation. Although a positive result is obtained in 2 separate runs without metabolic activation, the validity of these results is questionable since the occurrence of cytotoxicity is not well documented.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1993
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
All studies have been performed in compliance with the standard procedures of the US National Toxicology Program. However, no full reports are available form the NTP database and verly limited or no data is available about cytotoxicity by which the interpretation of the results on an individual basis is very difficult.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
induced male Syrian hamster liver S9 and induced male Sprague Dawley rat liver S9
Test concentrations with justification for top dose:
1 - 1000 ug/L depending upon strain
Vehicle / solvent:
95% Ethanol
Details on test system and experimental conditions:
In the standard protocol (preincubation) for conducting the Ames assay, a test tube containing a suspension of one strain of Salmonella typhimurium (or E. coli) plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical. Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are prepared; these contain the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen*. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow. These colonies are comprised of bacteria that have undergone reverse mutation to restore function of the histidine-manufacturing gene. The number of colonies is usually counted after 2 days.

Several modifications of the Ames test protocol have been used over the years in special circumstances. These include standard plate incorporation (no preincubation step prior to plating onto Petri dishes), FMN reduction (use of flavin mononucleotide for reduction of test articles such as azo dyes), plate test with volatile liquids (exposure of bacteria in a sealed Petri dish), cecal reduction (use of rat cecal bacteria to provide reduction of azo compounds), and plate tests conducted within a sealed dessicator (gas chamber) for exposure to gaseous substances. The specific test protocol that was used in an Ames test is noted in the description of the assay data.

Spontaneous mutations (those that occur by chance, not by chemical treatment) will appear as colonies on the control petri dishes. If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain of bacteria. The positive control plates are also counted, and the number of mutant colonies appearing on them must be significantly increased over the spontaneous control number for the test to be considered valid. Failure of the positive control chemical to induce mutation is reason to discard the experiment.

Several doses (usually at least 5) of each test chemical and multiple strains of bacteria are used in each experiment. In addition, cultures are set up with and without added liver S9 enzymes at varying concentrations. Therefore, a variety of culture conditions are employed to maximize the opportunity to detect a mutagenic chemical. In analyzing the data, the pattern and the strength of the mutant response are taken into account in determining the mutagenicity of a chemical. All observed responses are verified in repeat tests. If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be nonmutagenic in the Ames test.

*Positive control chemicals used in NTP Ames tests:

For strains tested in the absence of S9


TA98, 2-nitrofluorene or alternatively, TA98 and TA1538, 4-nitro-o-phenylenediamine
TA100 and TA1535, sodium azide
TA97 and TA1537, 9-aminoacridine
TA102, mitomycin C
TA104, methyl methanesulfonate
E.coli WP2 uvrA pKM101, methyl methanesulfonate

For strains tested with S9

All strains, 2-aminoanthracene (or occasionally, sterigmatocystin)
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

See original document with results

Conclusions:
Calcium Naphthenate was non-mutagenic in the Salmonella bacterial Mutagenicity test system.
Executive summary:

Calcium Naphthenate was non-mutagenic in the Salmonella bacterial Mutagenicity test system strains TA100, TA1535, TA97 and TA98 at 1 - 1000 ug/L depending upon strain wiht and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
See original document
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
E. coli WP2
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver S9
Test concentrations with justification for top dose:
31.25 - 62.5 - 125 - 250 - 500 - 1000 - 2000 - 4000 µg/plate
Vehicle / solvent:
Unspecified 'Carrier oil'
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

See original document

Conclusions:
Calcium Naphthenate was non-mutagenic in the E-coli and Salmonella bacterial reverse mutagenicity test system.
Executive summary:

Calcium Naphthenate was non-mutagenic in the E-coli and Salmonella bacterial reverse mutagenicity test system (strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100) when tested at 31.25 - 4000 µg/plate wiht and without metabolic activation.

Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)
Deviations:
not specified
Principles of method if other than guideline:
See original document
GLP compliance:
no
Type of assay:
yeast cytogenetic assay
Species / strain / cell type:
Saccharomyces cerevisiae
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver S9
Test concentrations with justification for top dose:
Exp. 1A without S9): 0 - 10 - 100 - 500 - 1000 - 2500 - 5000 µg/plate
Exp. 1B (with S9) : 0 - 10 - 100 - 500 - 1000 - 2500 - 5000 µg/plate
Exp. 2AB (without S9) : 0 - 10 - 100 - 500 - 1000 - 2500 - 5000 µg/plate
Exp. 2B (whit S9) : 0 - 10 - 100 - 500 - 1000 - 2500 - 5000 µg/plate
Vehicle / solvent:
Unspecified 'Carrier oil'
Species / strain:
Saccharomyces cerevisiae
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

See original document

Conclusions:
Calcium Naphthenate is non-mutagenic in the Yeast gene conversion test system.
Executive summary:

Calcium Naphthenate was non-mutagenic in the Yeast gene conversion test system Saccharomyces cerevisiae when tested at 10-5000 µg/plate with and without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
See original document
Only without metabolic activation
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
hepatocytes: Rat Liver RL4 cells
Metabolic activation:
without
Test concentrations with justification for top dose:
62.5 - 125 - 250 µg/ml
Vehicle / solvent:
Unspecified 'Carrier oil'
Species / strain:
mammalian cell line, other: Rat,Liver RL4
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

See original document

Conclusions:
Calcium Naphthenate is non-mutagenic in the Rat Liver chromosomal damage test system.
Executive summary:

Calcium Naphthenate is non-mutagenic in the Rat Liver chromosomal damage test system at 62.5-250 µg/ml without metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Rat, Liver, S-9, Aroclor 1254
Test concentrations with justification for top dose:
0.005 - 0.5 µg/mL
Vehicle / solvent:
DMSO and Acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
With S9
Details on test system and experimental conditions:
L5178Y T K +/- mouse lymphoma cells were originally obtained from Dr. Donald Clive, Burroughs Wellcome Co. (Research Triangle Park, NC). The cells were grown in Fischer's medium for leukemic cells of mice (Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI). Cells were screened for the presence of mycoplasma after cryopreservation. New cultures were initiated at approximately 3 month intervals from cells stored in liquid N 2. The toxicity of each chemical was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague—Dawley rats. S9 mix was prepared according to the procedure of Clive et al. (1). Cells at a concentration of 6 x 105/mL (6 x 106 cells total) were exposed for 4 h to a range of concentrations from 0.005 to 0.5 µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37 ± 1 °C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0—90% cytotoxicity. For each assay, there were 2—4 solvent controls, a positive control of ethyl methylsulfonate at 4.7 x 10~6 M (or methyl methanesulfonate at 10—2Q figl mL) for the test without metabolic activation, and a positive control of 3-methylcholanthrene at 1.86 x 10~5 M (or dimethylbenz[a]- anthracene at 0.5—4 ^g/mL) for the test with metabolic activation. The mutagenicity assay was performed according to the procedure described by Clive and Spector (2). A total of 1.2 x 107 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ± 1 °C, washed twice with growth medium, and maintained at 37 ± 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 x 105/mL at 24 h intervals. They were then cloned (1 x 106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar (BBL, Inc., Cockeysville, MD). Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 jxglmL) to the cloning medium for mutant selection. The lOOx stock solution of TFT in saline was stored at —70 °C and was thawed immediately before use. Plates were incubated at 37 ± 1 °C in 5% CO2 in air for 10—12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter (Synbiosis, Frederick, MD). Only colonies larger than ~0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 10 6 surviving cells. Although there are several different methods for evaluating mouse lymphoma data, results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect (1). Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from ~0.2 to 1.1 mm.

References:
1. Clive, D , Johnson, K O , Spector, J E S , Batson, A G , and Brown, M M M (1979) Validation and charactenzation of the L5178Y/TK-+/- mouse lymphoma mutagen assay system Mutat Res 59, 61 —108.
2. Clive, D, and Spector, J F S (1975) Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y TK-+/-— mouse lymphoma cells Mutat Res 31, 17—29.
Evaluation criteria:
Positive scores were given were the mutant frquency was more than doubling the values of the solvent treated controls, together with evidence of a dose related increase.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
other: Yes, DMSO
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
other: Yes, Aceton
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
other: Yes, Aceton
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Negative (CCRIS Record number: 1169; last revision date: 2010 -06-01)

Conclusions:
The original publication reports a postive genetoxic effect for calcium naphthenate with Acetone as solvent and in the abscence of metabolic activation. However when studying the raw data and the evaluation criteria the applicant can not support this conclusion. In stead absence of genotoxicity is concluded by the applicant.
Executive summary:

Mammalian mutagenicity of calcium naphtenate was studied in L5178Y T K +/-mouse lymphoma cells both with and without metabolic activation at 0.0005 to 10000 µg/mL. The original publication reports a postive genetoxic effect for calcium naphthenate with acetone as solvent and in the abscence of metabolic activation. However when studying the raw data and the evaluation criteria the applicant can not support this conclusion. In stead absence of genotoxicity is concluded by the applicant.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
Mutagenicity properties have been estimated using the validated VEGA modelling tool (Istituto di Ricerche Farmacologiche Mario Negri)
Qualifier:
no guideline followed
Principles of method if other than guideline:
The mutagenicity has been predicted using the VEGA mutagenicity model which is an extension of the original CAESAR model.
GLP compliance:
no
Remarks:
not applicable for QSARs
Type of assay:
other: QSAR
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Species / strain:
not specified
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
1.Defined endpoint: Mutagenicity: genetic toxicity in vitro
2. Unambiguous algorithm: The first model uses a trained support vector machine classifier to classify results into mutagenic or non-mutagenic classes. The second model is based on structural alerst. If the molecule is classified as non-mutagenic in the first model, it is tested for structural alerts in the second model. If there are structure alerts the molecule is classififed as suspicious mutagen.
3. Applicability domain: The applicability domain is indicated by a globa applicability domain index (ADI) which takes into account similar molecules with known experimental values, accuracy of prediction for similar molecules, concordance for similar molceuls, atom centered fragments similary check, model descriptors range chek. The ADI has values from 0 (worst case) to 1 (best case)
4. Statistical characteristics: training set: n = 3275, accuracy = 0.92, specificity = 0.87, sensitivity = 0.96
test set : n = 805, accuracy = 0.82, specificity= 0.74, sensitivity = 0.89
5. Mechanistic interpretation: no structural alerts.
Adequacy of prediction:
The naphtenic acids fall within the applicability domain described above: the ADI ranges from 1 to 0.915. Thus the VEGA model makes a reliable prediction of the mutagenicity of naphthenic acids.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Since naphthenic acids can adopt a wide variety of structures, qsars are peformed for a large number of different structures. The structures are chosen in order to represent the complete composition of the mixture of naphthenic acids.

The results of the different structures are given in the table below , along with ADI (Applicability Domain Index).

C-number Ring Branch SMILES Mw Predicted ADI
C6 - - C(=O)(O)CCCCC 116,16 neg 1
C7 - - C(=O)(O)CCCCCC 130,19 neg 1
C8 - - C(=O)(O)CCCCCCC 144,22  neg 1
C8 1 pentane - C(=O)(O)CCC1CCCC1 142,2 neg 0,975
C9 - methyl C(=O)(O)CCCC(C)CCC 158,24 neg 0,997
C10 - ethyl C(=O)(O)CCCC(CCC)CC 172,27 neg 0,998
C10 1 pentane - C(=O)(O)CCC1C(CC)CCC1 170,25 neg 0,976
C11 1 pentane - C(=O)(O)CCC1C(CCC)CCC1 184,28 neg 0,977
C12 1 hexane - C(=O)(O)CCC1C(CCC)CCCC1 198,31 neg 0,976
C12 1 pentane - C(=O)(O)CCC1C(CCCC)CCC1 198,31 neg 0,976
C12 2 pentanes fused - C(=O)(O)CCC1C2C(C)CCC2CC1 196,29 neg 0,921
C13 2 pentanes - C(=O)(O)CCC1C(C2CCCC2)CCC1 210,32 neg 0,931
C13 pentane hexane fused   O=C(O)CCC2CCCC1CCC(C)C12 210,32 neg 0,924
C14 1 hexane - O=C(O)C1C(CCCCCCCC)CCC1 226,36 neg 0,975
C14 2 pentanes - C(=O)(O)CCC1CC(C2C(C)CCC2)CC1 224,35 neg 0,931
C14 2 pentanes fused - C(=O)(O)CCCC1C2C(CC)CCC2CC1 224,35 neg 0,925
C15 - propyl C(=O)(O)CCCCC(CCCCCC)CCC 242,41 neg 0,997
C15 1 pentane ethyl C(=O)(O)CCC1C(CC(CCC)CC)CCC1 240,39 neg 0,975
C15 2 pentanes - C(=O)(O)CCC1C(CC2CC(C)CC2)CCC1 238,37 neg 0,93
C15 2 hexanes fused   O=C(O)CCC1CCC2CCCC(CC)C2C1 238,37 neg 0,927
C15 3 pentanes fused - C(=O)(O)CCCC1C2C3C(CC2CC1)CCC3C 250,38 neg 0,966
C16 1 pentane - C(=O)(O)CCC1C(CCCCCCCC)CCC1 254,42 neg 0,975
C16 2 pentanes - C(=O)(O)CCC1CC(C2C(CCC)CCC2)CC1 252,4 neg 0,93
C16 2 hexane - C(=O)(O)C1C(CCC2C(C)CCCC2)CCCC1 252,4 neg 0,93
C16 3 pentanes of which 2 fused - C(=O)(O)CC1C2C(CC3CCCC3)CCC2CC1 250,38 neg 0,948
C17 1 pentane - C(=O)(O)CCC1C(CCCCCCCCC)CCC1 268,44 neg 0,973
C17 4 pentanes fused - C12(C3C(CC(=O)O)CCC3CC1)C1C(CCC1)CC2 262,4 neg 0,97
C18 1 hexane propyl C(=O)(O)CCC1C(CC(CCCC)CCC)CCCC1 282,47 neg 0,972
C18 3 pentanes of which 2 fused - C(=O)(O)CCCCC1C2C(C3CCCC3)CCC2CC1 278,44 neg 0,955
C19 2 pentanes fused ethyl C(=O)(O)CCCCC1C2C(C(CCC)CC)CCC2CC1 294,48 neg 0,939
C19 2 pentane propyl C(=O)(O)CC1CC(CC(CCC2CCCC2)CCC)CC1 294,48 neg 0,927
C25 2 hexane propyl C(=O)(O)CCCC(CCC1C(CCC2C(C)CCCC2)CCCC1)CCC 378,64 neg 0,921
C30 2 hexane propyl C(=O)(O)CCC(CCCC1C(CCC2C(CCCCCC)CCCC2)CCCC1)CCC 448,78 neg 0,915
C30 3 hexane - C(=O)(O)CCCCCCC1C(CCC2C(CCCC3CCCCC3)CCCC2)CCCC1 446,76 neg 0,936
C30 3 hexanes fused ethyl-ethyl O=C(O)CCCCC(CC)CCC1CC2CCCC3CC(CCC(CC)CC)CC(C1)C23 446,76 neg 0,976
C30 2 hexanes fused 1 not propyl O=C(O)CCCC(CCC)CCCC1CCC2CC(CCC2C1)CC3CCCC(C)C3 432,74 neg 0,961
Conclusions:
The substance naphthenic acids was predicted to be non-mutagen for the various chain lengths.
Executive summary:

The substance naphthenic acids, including various molecules from C6 -C30 chain lengths, was predicted to be consistently non-mutagen using the VEGA QSAR model which is an extension of the original CAESAR model. If the prediction is confirmed by other results e.g. in a weight of evidence approach, this result can be used for classification and risk assessment.

Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: genome mutation
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Justification for type of information:
QSAR prediction
Guideline:
other: Guidance on information requirements and chemical safety assessment Chapter R.7a: Endpoint specific guidance, non-testing data on mutagenicity, p 380-381
Principles of method if other than guideline:
Toxtree: Benigni-Bossa rulebase for mutagenicity. This is an expert system, a decision tree based on structural alerts.
GLP compliance:
no
Remarks:
not applicable
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Test concentrations with justification for top dose:
not applicable
Vehicle / solvent:
not applicable
Details on test system and experimental conditions:
not applicable
Evaluation criteria:
not applicable
Statistics:
not applicable
Species / strain:
not specified
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
1.Defined endpoint: genotoxic carcinogenicity
2. Unambiguous algorithm: Expert system, decision tree based on structural alerts
3. Applicability domain: The applicability domain of each alert is defined by its modulating factors.
4. Statistical characteristics: sensitivity = 0.85, specifcity = 0.72, accuracy = 0.78
5. Mechanistic interpretation: The alerts of the model were derived from existing mechanistic knowledge. Since there is no structural alert, there is no mechanistic interpretation of this chemical.
Adequacy of prediction:
The prediction can be assumed to be adequate by considering the structure of the naphthenic acids. The molecule does not contain any structural group represented by the structural alerts. Since there are no alerts, modulating factors do not need to be taken into account and the prediction can be believed to be reliable.
Remarks on result:
other: all strains/cell types tested

Since naphthenic acids can adopt a wide variety of structures, QSARs are peformed for a large number of different structures. The structures are chosen in order to represent the complete composition of the mixture of naphthenic acids.

The results of the different structures are given in the table.

C-No. Ring Branch SMILES MW predicted
C6 - - C(=O)(O)CCCCC 116,16 neg
C7 - - C(=O)(O)CCCCCC 130,19 neg
C8 - - C(=O)(O)CCCCCCC 144,22  neg
C8 1 pentane - C(=O)(O)CCC1CCCC1 142,2 neg
C9 - methyl C(=O)(O)CCCC(C)CCC 158,24 neg
C10 - ethyl C(=O)(O)CCCC(CCC)CC 172,27 neg
C10 1 pentane - C(=O)(O)CCC1C(CC)CCC1 170,25 neg
C11 1 pentane - C(=O)(O)CCC1C(CCC)CCC1 184,28 neg
C12 1 hexane - C(=O)(O)CCC1C(CCC)CCCC1 198,31 neg
C12 1 pentane - C(=O)(O)CCC1C(CCCC)CCC1 198,31 neg
C12 2 pentanes fused - C(=O)(O)CCC1C2C(C)CCC2CC1 196,29 neg
C13 2 pentanes - C(=O)(O)CCC1C(C2CCCC2)CCC1 210,32 neg
C13 pentane hexane fused   O=C(O)CCC2CCCC1CCC(C)C12 210,32 neg
C14 1 hexane - O=C(O)C1C(CCCCCCCC)CCC1 226,36 neg
C14 2 pentanes - C(=O)(O)CCC1CC(C2C(C)CCC2)CC1 224,35 neg
C14 2 pentanes fused - C(=O)(O)CCCC1C2C(CC)CCC2CC1 224,35 neg
C15 - propyl C(=O)(O)CCCCC(CCCCCC)CCC 242,41 neg
C15 1 pentane ethyl C(=O)(O)CCC1C(CC(CCC)CC)CCC1 240,39 neg
C15 2 pentanes - C(=O)(O)CCC1C(CC2CC(C)CC2)CCC1 238,37 neg
C15 2 hexanes fused   O=C(O)CCC1CCC2CCCC(CC)C2C1 238,37 neg
C15 3 pentanes fused - C(=O)(O)CCCC1C2C3C(CC2CC1)CCC3C 250,38 neg
C16 1 pentane - C(=O)(O)CCC1C(CCCCCCCC)CCC1 254,42 neg
C16 2 pentanes - C(=O)(O)CCC1CC(C2C(CCC)CCC2)CC1 252,4 neg
C16 2 hexane - C(=O)(O)C1C(CCC2C(C)CCCC2)CCCC1 252,4 neg
C16 3 pentanes of which 2 fused - C(=O)(O)CC1C2C(CC3CCCC3)CCC2CC1 250,38 neg
C17 1 pentane - C(=O)(O)CCC1C(CCCCCCCCC)CCC1 268,44 neg
C17 4 pentanes fused - C12(C3C(CC(=O)O)CCC3CC1)C1C(CCC1)CC2 262,4 neg
C18 1 hexane propyl C(=O)(O)CCC1C(CC(CCCC)CCC)CCCC1 282,47 neg
C18 3 pentanes of which 2 fused - C(=O)(O)CCCCC1C2C(C3CCCC3)CCC2CC1 278,44 neg
C19 2 pentanes fused ethyl C(=O)(O)CCCCC1C2C(C(CCC)CC)CCC2CC1 294,48 neg
C19 2 pentane propyl C(=O)(O)CC1CC(CC(CCC2CCCC2)CCC)CC1 294,48 neg
C25 2 hexane propyl C(=O)(O)CCCC(CCC1C(CCC2C(C)CCCC2)CCCC1)CCC 378,64 neg
C30 2 hexane propyl C(=O)(O)CCC(CCCC1C(CCC2C(CCCCCC)CCCC2)CCCC1)CCC 448,78 neg
C30 3 hexane - C(=O)(O)CCCCCCC1C(CCC2C(CCCC3CCCCC3)CCCC2)CCCC1 446,76 neg
C30 3 hexanes fused ethyl-ethyl O=C(O)CCCCC(CC)CCC1CC2CCCC3CC(CCC(CC)CC)CC(C1)C23 446,76 neg
C30 2 hexanes fused 1 not propyl O=C(O)CCCC(CCC)CCCC1CCC2CC(CCC2C1)CC3CCCC(C)C3 432,74 neg
Conclusions:
Considering the QSAR Toxtree: Benigni-Bossa rulebase for mutagenicity, the substance naphthenic acids was predicted to be non-mutagen.
Executive summary:

The substance Naphthenic acids, including various molecules from C6 -C30 chain lengths, was predicted to be consistently non-mutagen using the Toxtree (Benigni-Bossa) rulebase for mutagenicity.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Available data on both sodium naphthenate and calcium naphthenate showed that these compounds were not genotoxic nor mutagenic in various in vitro test systems. Since in both molecules the naphthenate ion is considered as the toxic entity a read across of the properties of genetic toxicity from these compounds to naphthenic acid is justified. Moreover these finding were further supported by the QSAR modelling of selected representative naphthenic acids, which were consistently predicted non-mutagenic. Finally, an in vivo Micronucleus assay with refined Naphthenic acids was negative.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is a GLP study and is recently (2010) performed according to standard methods, there it is considered reliable, adequate and relevant.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Principles of method if other than guideline:
The micronucleus test was consistent with the US EPA guidelines for studies of this type (OPPTS 870.5395) and with OECD 474. The testing was in accordance with Good Laboratory Practice Guidelines of the OECD (OECD, 1997) and the U.S. EPA (CFR, 2007). The study was an extension of the combined repeated dose/reproductive and developmental toxicity screening in rats.

Bone marrow was collected from all animals at terminal sacrifice and flushed into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). The bone marrow was centrifuged, the majority of the HI FBS was decanted, and the pellet was re-suspended. Bone marrow smears were prepared by placing single drops of suspension on microscope slides (minimum of two per preparation). The slides were coded, air dried, fixed in methanol and allowed to air dry a second time.

Coded slides were stained with acridine orange (Hayashi et al., 1983). A total of 1000 erythrocytes/slide were evaluated (both polychromatic (PCE) and normochromatic erythrocytes (NCE)), and the PCE/NCE ratio was calculated. The number of micronucleated PCEs from a total of 2000 PCEs was then determined for each animal.

The percentages of PCEs , micronucleated cells in NCEs, and the ratios of PCEs to total erythrocytes in the test substance- and vehicle-treated groups were compared using ANOV A (Snedecor and Cochran, 1980). If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett Test (Dunnett, 1964) was used to compare each test substance-treated group to the vehicle control group. In addition, the positive control and vehicle control groups were compared using a separate parametric one-way ANOVA (Snedecor and Cochran, 1980).
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
no data
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: no data

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data
Duration of treatment / exposure:

Approximately 30 days
Frequency of treatment:
Daily
Post exposure period:
None
Remarks:
Doses / Concentrations:
0, 100, 300, 900 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
6 males/6 females
Control animals:
yes, concurrent vehicle
yes, sham-exposed
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: oral gavage
- Doses / concentrations: 60 mg/kg/day
Tissues and cell types examined:
bone marrow , polychromatic and normochromatic eryhtrocytes
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
Bone marrow was collected from all animals at terminal sacrifice

DETAILS OF SLIDE PREPARATION:
Bone marrow was collected from all animals at terminal sacrifice and flushed into a centrifuge tube using a syringe containing heat inactivated fetal bovine serum (HI FBS). The bone marrow was centrifuged, the majority of the HI FBS was decanted, and the pellet was re-suspended. Bone marrow smears were prepared by placing single drops of suspension on microscope slides (minimum of two per preparation). The slides were coded, air dried, fixed in methanol and allowed to air dry a second time. Coded slides were stained with acridine orange (Hayashi et al., 1983).

METHOD OF ANALYSIS:
A total of 1000 erythrocytes/slide were evaluated (both polychromatic (PCE) and normochromatic erythrocytes (NCE)), and the PCE/NCE ratio was calculated. The number of micronucleated PCEs from a total of 2000 PCEs was then determined for each animal.

The percentages of PCEs , micronucleated cells in NCEs, and the ratios of PCEs to total erythrocytes in the test substance- and vehicle-treated groups were compared using ANOV A (Snedecor and Cochran, 1980). If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett Test (Dunnett, 1964) was used to compare each test substance-treated group to the vehicle control group. In addition, the positive control and vehicle control groups were compared using a separate parametric one-way ANOVA (Snedecor and Cochran, 1980).
Statistics:
The percentages of PCEs , micronucleated cells in NCEs, and the ratios of PCEs to total erythrocytes in the test substance- and vehicle-treated groups were compared using ANOV A (Snedecor and Cochran, 1980). If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett Test (Dunnett, 1964) was used to compare each test substance-treated group to the vehicle control group. In addition, the positive control and vehicle control groups were compared using a separate parametric one-way ANOVA (Snedecor and Cochran, 1980).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Systemic toxicity: No treatment-related bone marrow effects.
Genotoxic effects: Not mutagenic. The frequencies of micronuclei in in bone marrow from rats treated with refined naphthenic acids did not differ statistically from those in the sham and vehicle control groups. A significant increase in micronucleus frequency was found in material harvested from rats treated with the positive control, cyclophosphamide providing evidence that the test had worked as expected.

Table 1. Summary of results of micronucleus data from rats following repeated treatment with refined naphthenic acids. 

Treatment

Gender

Total MN PCEs/2000 PCEs (N=5)

% MN PCEs

Total MN NCEs/2000 NCEs (N=5)

Ratio of PCEs to Total Erythrocytes

Corn Oil

Males

8

0.08+0.08

3

0.54+0.07

 

Females

8

0.08+0.12

4

0.69+0.11

 

 

 

 

 

 

Sham Control

Males

6

0.06+0.04

3

0.52+0.11

 

Females

8

0.08+0.08

2

0.55+0.17

 

 

 

 

 

 

Naphthenic Acid

 

 

 

 

 

100 mg/kg/day

Males

7

0.07+0.07

1

0.53+0.09

 

Females

4

0.04+0.04

7

0.65+0.16

300 mg/kg/day

Males

4

0.04+0.04

3

0.49+0.67

 

Females

5

0.06+0.05

5

0.67+0.13

900 mg/kg/day

Males

8

0.08+0.08

5

0.61+0.11

 

Females

5

0.06+0.05

5

0.75+0.19

 

 

 

 

 

 

Positive Control (Cyclophosphamide)

60 mg/kg/day

Males

128

1.28+0.14a

13

0.40+0.21

 

Females

97

0.97+0.19a

16

0.51+0.12a

Conclusions:
Interpretation of results: negative
Naphthenic acids did not induce chromosomal aberrations under the conditions of the test
Executive summary:

A micronucleus test was conducted in male and female Wistar rats with refined Naphtenic acids dosed at 100, 300 and 900 mg/kg bw by oral gavage.A total of 1000 erythrocytes/slide were evaluated (both polychromatic PCE and normochromatic erythrocytes NCE), and the PCE/NCE ratio was calculated. The number of micronucleated PCEs from a total of 2000 PCEs was then determined for each animal.The frequencies of micronuclei in in bone marrow did not differ statistically from those in the sham and vehicle control groups. A significant increase in micronucleus frequency was found in material harvested from rats treated with the positive control, cyclophosphamide providing evidence that the test had worked as expected.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

No experimental in vitro genetic toxicity studies were available for Naphthenic acids, however Weight of Evidence was available from ‘Sodium naphthenates’:

- Salmonella bacterial mutagenicity: negative up to >333 μg/L with and without metabolic activation (NTP, 1993; HPVIS, 2012).

- Chromosomal aberration: negative in CHO cells at 54, 116 & 250 μg/mL without metabolic activation and 25, 54, 116 & 250 μg/mL with metabolic activation (NTP, 1993; HPVIS, 2012).

- Sister Chromatid Exchange: weakly positive to positive when tested at concentrations of 17, 59, 167, 500 ug/mL (Trial 1; without metabolic activation) and 100, 150, 200, 250 µg/mL (Trial 2; without metabolic activation) and negative at 17, 59, 167, 500 µg/mL with metabolic activation (NTP, 1993; HPVIS, 2012). Although a positive result is obtained in 2 separate runs without metabolic activation, the validity of these results is questionable since the occurrence of cytotoxicity is not well documented.

 

Further Weight of Evidence is available from ‘Calcium naphthenates’:

- E-coli and Salmonella bacterial reverse mutagenicity: negative in WP2 uvr A and Salmonella TA 1535, TA 1537, TA 98 and TA 100 strains when tested at 31.25 - 4000 μg/plate with and without metabolic activation (Shell Research Ltd, 1983).

- Saccharomyces cerevisiae: non-mutagenic when tested at 10 -5000 μg/plate with and without metabolic activation (Shell Research Ltd, 1983).

- Rat Liver chromosomal damage: non-mutagenic at 62.5-250 μg/mL without metabolic activation (Shell Research Ltd, 1983).

- In vitro testing in L5178Y T K +/-mouse lymphoma cells both with and without metabolic activation at 0.0005 to 10000 µg/mL showed a positive effect in the absence of metabolic activation (Seifried et al, 2006), however when studying the raw data and the evaluation criteria the applicant can not support this conclusion.

 

Additional Weight of Evidence is available from QSAR prediction on the various molecules (C6 -C30 chain lengths):

- VEGA QSAR model which is an extension of the original CAESAR model (Ferrari & Gini, 2010; Benigni et al;, 2008): consistently non-mutagenic.

- Toxtree: Benigni-Bossa rulebase for mutagenicity (Benigni et al., 2008; Benigni et al., 2007): consistently non-mutagenic.

 

Finally, an in vivo Micronucleus test was conducted in male and female Wistar rats with refined Naphthenic acids dosed at 100, 300 and 900 mg/kg bw by oral gavage (HPVIS, 2010). A total of 1000 erythrocytes/slide were evaluated (both polychromatic PCE and normochromatic erythrocytes NCE), and the PCE/NCE ratio was calculated. The number of micronucleated PCEs from a total of 2000 PCEs was then determined for each animal.The frequencies of micronuclei in in bone marrow did not differ statistically from those in the sham and vehicle control groups. A significant increase in micronucleus frequency was found in material harvested from rats treated with the positive control, cyclophosphamide providing evidence that the test had worked as expected.


Justification for selection of genetic toxicity endpoint
Although an extensive battery of in vitro and in silico (QSAR) data were available as Weight of Evidence, the key study was an in vivo Micronucleus assay.

Justification for classification or non-classification

All genotoxicity and mutagenicity tests and modelling tools revealed the absence of genotoxic and mutagenic properties for naphthenic acids. Therefore a non-classification for this endpoint is justified.