Naphthenic acids, lithium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are no data for lithium naphthenate. Data from appropriate read across substances are presented, with data on fatty acids C18 (unsaturated) lithium salts, sodium naphthenate, calcium naphthenate and naphthenic acids.

 

For fatty acids C18 (unsaturated) lithium salts, the in vitro gene mutation (bacterial reversion assay/Ames test) was negative, the in vitro gene mutation (mammalian cell TK ± assay in L5178Y mouse lymphoma cells) was negative, and the in vitro cytogenicity (chromosomal aberration assay in human lymphocytes) was negative.

 

Calcium and sodium naphthenate were not mutagenic in bacterial reverse mutation (Ames) assays conducted (according to the NTP test protocol) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 in the presence and absence of mammalian metabolic activation (S9) (National Toxicology Program, 1993a,b). Calcium naphthenate showed a similar lack of mutagenic activity in a non-guideline investigation with strains of S. typhimurium and Escherichia coli (Shell Research Ltd, 1983).

 

In a mouse lymphoma assay, evidence of mutagenic potential was reported when calcium naphthenate was tested without S9 with acetone as the vehicle (Seifried et al., 2006). However, when studying the raw data and the evaluation criteria, this conclusion cannot be supported. Instead, a lack of genotoxicity is concluded.

 

Sodium naphthenate was not clastogenic in Chinese hamster ovary (CHO) cells, when tested with and without S9 (National Toxicology Program, 1993c). Calcium naphthenate showed a similar lack of clastogenic activity in a non-guideline study with rat liver RL4 cells when tested in the absence of S9 (Shell Research Ltd, 1983).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 01 February 2010 and 22 February 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

Please see the Read Across Justification Documents in Section 13 of IUCLID dossier.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment one: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone.

- Justification for choice of solvent/vehicle: The test material was insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in–house. Sterile distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested that the test material was insoluble in water. The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix. Benzo(a)pyrene: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 mix. 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix. 9-Aminoacridine: 80 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix. N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix. N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix. N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) - Experiment 1

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Example:
METHOD OF APPLICATION: in agar (pre-incubation) - Experiment 2

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): 20 minutes at 37 degrees C

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10(+09) bacteria per mL.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation
Dunnetts Linear Regression Analysis

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Remarks:

The test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Example:
TEST-SPECIFIC CONFOUNDING FACTORS
- Vehicle solubility: The test material was insoluble in dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in–house. Sterile distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested that the test material was insoluble in water. The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle.
- Precipitation: A greasy precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was toxic at and above 1500 µg/plate to TA100 without S9 and WP2uvrA- with S9 and was non-toxic to TA100 with S9 and WP2uvrA- without S9. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number. However, the toxicity of the test material to the tester strains was of insufficient severity to prevent testing up to the maximum recommended dose level of 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

RESULTS

Preliminary Toxicity Test

The test material was toxic at and above 1500 µg/plate to TA100 without S9 and WP2uvrA^-with S9 and was non-toxic to TA100 with S9 and WP2uvrA^-without S9. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000

-	TA100	95	106	100	101	104	103	107	112	99	67 *	0 P *
+	TA100	101	86	97	92	102	93	107	93	95	70	75P
-	WP2uvrA^-	21	25	23	20	20	19	20	21	20	21	13P
+	WP2uvrA^-	32	26	20	29	20	30	21	31	19	56 *	60 P *

P         Precipitate

*          Partial or complete absence of bacterial background lawn

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented inTable1and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 1 (see attached document).

A history profile of vehicle and positive control values is presented in Appendix 4 (see attached document).

The test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number. However, the toxicity of the test material to the tester strains was of insufficient severity to prevent testing up to the maximum recommended dose level of 5000 µg/plate. A greasy precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1               Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test

Number of revertants (mean number of colonies per plate) Base-pair substitution type Frameshift type TA100 TA1535 WP2uvrA^- TA98 TA1537 109   25   20   18   11   97 (99) 26 (25) 20 (19) 14 (18) 11 (12) 91   25   16   22   14

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
94		21		20		17		4
107	(98)	16	(18)	22	(21)	21	(19)	13	(8)
94		17		21		20		8

 

Table 2               Test Results: Range-Finding Test– Without Metabolic Activation

Test period		From: 14 February 2010					To: 17 February 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98			TA1537
-	0	103 110 90	(101) 10.1#	25 24 19	(23) 3.2	27 27 30		(28) 1.7	20 18 15		(18) 2.5	16 14 13		(14) 1.5
-	5	102 128 128	(119) 15.0	26 24 24	(25) 1.2	19 25 31		(25) 6.0	14 19 19		(17) 2.9	14 12 14		(13) 1.2
-	15	111 81 124	(105) 22.1	22 19 20	(20) 1.5	21 21 23		(22) 1.2	16 15 18		(16) 1.5	8 19 16		(14) 5.7
-	50	107 130 128	(122) 12.7	19 24 14	(19) 5.0	31 23 18		(24) 6.6	18 13 13		(15) 2.9	15 13 20		(16) 3.6
-	150	123 109 129	(120) 10.3	12 14 22	(16) 5.3	16 29 29		(25) 7.5	12 15 16		(14) 2.1	10 13 10		(11) 1.7
-	500	100 95 108	(101) 6.6	12 15 23	(17) 5.7	23 25 30		(26) 3.6	19 15 15		(16) 2.3	8 12 11		(10) 2.1
-	1500	85 P 79 P 91 P	(85) 6.0	15 P 15 P 14 P	(15) 0.6	18 P 19 P 22 P		(20) 2.1	15 P 18 P 19 P		(17) 2.1	4 P 3 P 5 P		(4) 1.0
-	5000	81 P * 95 P * 91 P *	(89) 7.2	16 P * 11 P * 14 P *	(14) 2.5	22 P 21 P 24 P		(22) 1.5	22 P * 11 P * 15 P *		(16) 5.6	12 P * 8 P * 5 P *		(8) 3.5
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO			9AA
		3		5		2			0.2			80
		411 408 420	(413) 6.2	2183 1544 2281	(2003) 400.2	866 872 883		(874) 8.6	124 122 134	(127) 6.4		618 1336 1015	(990) 359.7

P        Precipitate

*         Partial absence of bacterial background lawn

#        Standard deviation

Table 3               Test Results: Range-Finding Test– With Metabolic Activation

Test period		From: 14 February 2010					To: 17 February 2010
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^‑			TA98		TA1537
+	0	98 96 96	(97) 1.2#	9 13 12	(11) 2.1	20 24 22		(22) 2.0	19 32 21	(24) 7.0	12 11 14	(12) 1.5
+	5	89 81 91	(87) 5.3	8 9 9	(9) 0.6	19 26 31		(25) 6.0	15 19 18	(17) 2.1	10 11 9	(10) 1.0
+	15	99 97 89	(95) 5.3	8 8 15	(10) 4.0	20 20 24		(21) 2.3	23 24 21	(23) 1.5	13 9 12	(11) 2.1
+	50	95 106 117	(106) 11.0	10 8 12	(10) 2.0	24 20 32		(25) 6.1	19 18 18	(18) 0.6	15 9 15	(13) 3.5
+	150	98 96 89	(94) 4.7	8 9 11	(9) 1.5	15 30 29		(25) 8.4	16 25 25	(22) 5.2	8 9 7	(8) 1.0
+	500	90 73 85	(83) 8.7	11 11 13	(12) 1.2	21 27 20		(23) 3.8	19 15 16	(17) 2.1	8 12 10	(10) 2.0
+	1500	82 P 81 P 84 P	(82) 1.5	8 P 13 P 8 P	(10) 2.9	19 P 31 P 23 P		(24) 6.1	12 P 10 P 19 P	(14) 4.7	9 P 15 P 11 P	(12) 3.1
+	5000	86 P * 97 P * 89 P *	(91) 5.7	12 P 12 P 11 P	(12) 0.6	20 P 22 P 23 P		(22) 1.5	15 P * 10 P * 19 P *	(15) 4.5	14 P 10 P 9 P	(11) 2.6
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		370 409 403	(394) 21.0	219 236 242	(232) 11.9	172 200 201		(191) 16.5	66 90 95	(84) 15.5	176 195 207	(193) 15.6

P        Precipitate

*         Partial absence of bacterial background lawn

#        Standard deviation

PLEASE SEE ATTACHED IN OVERALL REMARKS, ATTACHMENTS 1) Tables 4 & 5 (Main Test)

2) Report of Results in Mutagenicity Test using Micro-organisms

Conclusions:

Interpretation of results: Negative
The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA^-were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels (5 and 15 µg/plate) and an expanded dose range were selected in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

Results.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn to several of the tester strains, at 5000 µg/plate. The presence of toxicity varied depending on strain type, exposure to S9 mix and experiment number. However, the toxicity of the test material to the tester strains was of insufficient severity to prevent testing up to the maximum recommended dose level of 5000 µg/plate. A greasy precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

02 February 2006 and 23 August 2006.med between 12 May 2008 and 28 August 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

See IUCLID section 13 for read across justification

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitabilityThe volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary toxicity test:
0, 19.5, 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml

Chromosome Aberration Test - Experiment 1: The final concentration of fatty acids C18 (unsaturated) lithium salts (µg/ml) in the controls and treatments of the 4(20)-hour without S9 group were 0*, 20, 40, 80, 160*, 240*, 320*, MMC 0.4* and for the 4(20)-hour with S9 group were 0*, 40, 80*, 160*, 240*, 320, 480, CP 5* (* = Dose levels selected for metaphase analysis).
Chromosome Aberration Test - Experiment 2: The final concentration of fatty acids C18 (unsaturated) lithium salts (µg/ml) in the controls and treatments of the 24-hour without S9 group were 0*, 20, 40, 80*, 120*, 160*, 240*, MMC 0.2* and for the 4(20)-hour with S9 group were 0*, 40, 80*, 160*, 240*, 320, 480, CP 5*(* = Dose levels selected for metaphase analysis).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not stated in report

Untreated negative controls:

yes

Remarks:

0 µg/ml

Negative solvent / vehicle controls:

yes

Remarks:

solvent treatment groups were used as the vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 at 5 µg/mL in both experiments

Untreated negative controls:

yes

Remarks:

0 µg/ml

Negative solvent / vehicle controls:

yes

Remarks:

solvent treatment groups were used as the vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 used at 0.4 and 0.2 µg/mL in both experiments.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in medium

DURATION
- Preincubation period: 48 hrs

- Exposure duration: Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent): Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs.

SELECTION AGENT (mutation assays): - not applicable

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED: 100/culture


DETERMINATION OF CYTOTOXICITY
- Method:
- Mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there was approximately 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy: Frequency of polyploid cells

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exhibited a very steep dose-response curve

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.

- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.

- Evaporation from medium: Not applicable.

- Water solubility: Not applicable, test material suspended in MEM

- Precipitation:
Preliminary toxicity test: A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 625-1250 µg/ml, in the 4(20)-hour pulse exposure groups and at and above 78.1 µg/ml in the continuous exposure group.

Chromosome aberration Test - Experiments 1 and 2: Precipitate observations taken in the Preliminary Toxicity Test were considered to be representative for the study.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test
Haemolysis of the red blood cells was observed at and above 312.5 µg/ml in all three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to156.25 and 312.5 µg/ml in the 4(20)-hour exposures in the absence and presence of metabolic activation (S9) respectively. The maximum dose with metaphases present in the 24-hour continuous exposure was 156.25 µg/ml. The test material induced clear evidence of toxicity in all of the exposure groups and the toxic dose response curve was very steep.
The selection of the maximum dose level was based on toxicity for the all of the exposure groups.


COMPARISON WITH HISTORICAL CONTROL DATA:
All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.


ADDITIONAL INFORMATION ON CYTOTOXICITY:

EXPERIMENT 1: The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were scorable metaphases present at 320 µg/ml in the absence of metabolic activation (S9). In the presence of S9 the maximum dose level of the test material with scorable metaphases was 240 µg/ml.


EXPERIMENT 2:
The qualitative assessment of the slides determined that there were scorable metaphases present at the maximum test material dose level of 240 µg/ml in both the absence and presence of S9.

Remarks on result:

other: all strains/cell types tested

Chromosome Aberration Test - Experiment 1

Mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 38% mitotic inhibition was achieved at 320 µg/ml in the absence of S9. In the presence of S9 only 18% mitotic inhibition was achieved at 240 µg/ml but complete mitotic inhibition was observed at 320 and 480 µg/ml.

The maximum dose level selected for metaphase analysis was the maximum dose level in each case where scorable metaphase cells were evident.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

 Chromosome Aberration Test - Experiment 2

Mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed and that 63% mitotic inhibition was achieved at 240 µg/ml in the absence of S9. In the presence of S9 22% mitotic inhibition was achieved at 240 µg/ml and total inhibition at the next dose level of 320 µg/ml. The maximum dose level selected for metaphase analysis was 240 µg/ml in both exposure groups.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.

The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Conclusions:

Interpretation of results: Negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction. 

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 2000/32/EC. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. 

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three or four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

Results. 

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments. The test material was toxic and exhibited a very steep dose-response curve. The dose range used included a dose level that induced approximately 50% mitotic inhibition or was the maximum sub-toxic dose level.

Conclusion. 

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

2006

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Article gives detailed overview of methodolgy applied and raw data are avaialble as annexes to the publication. Although in the article the conclusions stated in tavtable 1 do not correspond with the raw data.

Justification for type of information:

See IUCLID section 13 for read across justification

Reason / purpose for cross-reference:

read-across source

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:

Interpretation of results (migrated information):
negative (Aceton)

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.

Reference 4

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Full study report available with very detailed infromation, although no GLP study. Major deficiency is the non-disclosure of the composition and identity of hte 'carrier oil'.

Justification for type of information:

See IUCLID section 13 for read across justification

Reason / purpose for cross-reference:

read-across source

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:

Interpretation of results (migrated information):
negative

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.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 19 April 2010 and 21 June 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

See IUCLID section 13 for read across justification

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity.

Vehicle and positive controls were used in parallel with the test material. Solvent (Acetone) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS), Sigma batch 0001423147 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

Vehicle used:
Vehicle (Acetone) treatment groups were used as the vehicle controls.

Justification for choice of vehicle:
Formed a suspension suitable for dosing at the required concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (Acetone) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (Acetone) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

This study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al, 1980; Jotz and Mitchell, 1981).

The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 and the United Kingdom Environmental Mutagen Society. The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).

Evaluation criteria:

Please see "Any other information on materials and methods incl. tables" section.

Statistics:

Please see "Any other information on materials and methods incl. tables" section.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS

Preliminary Toxicity Test

The dose range of the test material used in the Preliminary Toxicity Test was 4.88 to 1250 µg/ml. In all three of the exposure groups there were marked reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve was very steep in all three of the exposure groups. A precipitate of the test material was observed at and above 39.06 µg/ml in the 4-hour exposure groups, and at and above 156.25 µg/ml in the 24 hour exposure group. In the subsequent mutagenicity test the maximum dose was limited by test material-induced toxicity.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

As was seen previously, there was evidence of marked toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 3 and 6). The levels of toxicity observed were very similar to those of the Preliminary Toxicity Test. There was no evidence of any significant reductions in viability (%V), therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. Optimum levels of toxicity were achieved in the presence of metabolic activation (Table 6). Optimum or near optimum levels of toxicity were not achieved in the absence of metabolic activation, despite using a narrow dose interval, due to the very sharp onset of test material-induced toxicity (Table 3). However, it was considered that, with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including a dose level that achieved the optimum level of toxicity in the presence of metabolic activation, or in the second experiment where optimum levels of toxicity were achieved in the absence of metabolic activation, the test material had been adequately tested. The excessive levels of toxicity observed at and above 60 µg/ml in the absence of metabolic activation, and at and above 100 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). The precipitate observations varied very slightly from those of the Preliminary Toxicity Test with a precipitate of test material observed at and above 20 µg/ml in the absence of metabolic activation, and at and above 80 µg/ml in the presence of metabolic activation. However, the purpose and integrity of the study was considered unaffected.

The numbers of small and large colonies and their analysis are presented in attached Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen previously, there was evidence of marked toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 9 and 12). The levels of toxicity observed in the 24 hour exposure group in the absence of metabolic activation were very similar to those of the Preliminary Toxicity Test with optimum levels of toxicity being achieved (Table 9). There was also evidence of a very modest reduction in viability (%V) at the upper surviving dose level in the absence of metabolic activation, indicating some residual toxicity may have occurred at this dose level. However, the lowering of the S9 concentration to 1% in this second experiment resulted in a much steeper toxicity curve being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1 and optimum or near optimum levels of toxicity were not achieved despite using a very narrow dose interval of 5 µg/ml (Table 12). Due to the nature of the toxicity exhibited by the test material it was considered that to achieve optimum toxicity in the presence of 1% metabolic activation would be incredibly difficult. Therefore, with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels in either the first or second experiment, including dose levels that induced optimum levels of toxicity, it was considered that the test material had been adequately tested. The excessive levels of toxicity observed at and above 70 µg/ml in the absence of metabolic activation, and at 70 µg/ml in the presence of metabolic activation, resulted in these dose levels not being plated for viability or TFT resistance. Both positive controls induced acceptable levels of toxicity (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had no effect on the toxicity of the test material.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). Precipitate of test material was not observed at any of the dose levels in either the absence or presence of metabolic activation.

The numbers of small and large colonies and their analysis are presented in attached Tables 10 and 13.

Remarks on result:

other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Remarks:

Migrated from field 'Test system'.

Please see Attached "Tables 1 to 13"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:

Interpretation of results: Non-mutagenic
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Methods. 

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (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 up to ten 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 of test material was selected following the results of a preliminary toxicity test and for Experiment 1 was 2.5 to 80 µg/ml in the absence of metabolic activation, and 10 to 120 µg/ml in the presence of metabolic activation. The test material dose range for Experiment 2 was 10 to 80 µg/ml in the absence of metabolic activation, and 5 to 70 µg/ml in the presence of metabolic activation.

Results. 

The maximum dose level used in the mutagenicity test was limited by test material-induced toxicity. Precipitate of test material was observed in Experiment 1, at and above 20 µg/ml in the absence of metabolic activation, and at and above 80 µg/ml in the presence of metabolic activation. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

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.

Conclusion. 

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

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.

Justification for type of information:

See IUCLID section 13 for read across justification

Reason / purpose for cross-reference:

read-across source

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:

Interpretation of results (migrated information):
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 conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are no data for lithium naphthenate. Data from appropriate read across substances are presented, with data on fatty acids C18 (unsaturated) lithium salts, sodium naphthenate, calcium naphthenate and naphthenic acids.

 

A key bacterial reversion assays (Ames test), a chromosomal aberration assay and a mouse lymphoma assay have been conducted on fatty acids C18 (unsaturated) lithium salts. In all cases the results were negative. It is appropriate therefore to read across the negative genotoxicity data on fatty acids C18 (unsaturated) lithium salts to lithium naphthenate, therefore lithium naphthenate is considered negative for genetic toxicity.

 

Furthermore, the mutagenic potential of calcium and sodium naphthenate was assessed in two in vitro bacterial reverse mutation (Ames) assays, conducted according to the NTP test protocol (equivalent to OECD Test Guideline 471). S. typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to calcium and sodium naphthenate (in ethanol) at respective concentrations of 1-1000 and 1-3333 μg/L (strain specific), both in the presence and absence of metabolic activation by mammalian liver S9 fraction, alongside appropriate vehicle and positive controls. No evidence of cytotoxicity or mutagenic activity was observed in either test (National Toxicology Program, 1993a,b). A similar non-guideline investigation was conducted on calcium naphthenate (in an unspecified ‘carrier oil’) using the same four S. typhimurium strains in addition to E. coli strains WP2 and WP2uvrA. The test material was not mutagenic when tested at 31.25-4000 μg/plate both with and without S9. As part of the same investigation, calcium naphthenate was assessed for its potential to induce mutations to Saccharomyces cerevisiae (yeast) in an assay equivalent to OECD Test Guideline 480. Calcium naphthenate was not mutagenic at test concentrations of 10-5000 μg/plate both with and without S9 (Shell Research Ltd, 1983).

 

The in vitro bacterial mutagenic potential of naphthenic acids (including various molecules from C6-C30 chain lengths) was further analysed using two (Q)SAR models. The substances were consistently predicted to be non-mutagenic in the VEGA model (an extension of the original CAESAR model) and in Toxtree (Benigni-Bossa rulebase) (Ferrari and Gini, 2010; Benigni et al., 2007).

 

The mammalian mutagenicity potential of calcium naphthenate was studied using mouse lymphoma L5178Y cells, in an assay equivalent to OECD Test Guideline 476. Cells were exposed to test material concentrations of 0.005-0.5 μg/mL (in acetone and DMSO) both in the presence and absence of S9 metabolic activation. Evidence of mutagenic potential was reported for calcium naphthenate when tested without S9 with acetone as the vehicle (Seifried et al., 2006). However, when studying the raw data and the evaluation criteria this conclusion cannot be supported. Instead, a lack of genotoxicity is concluded.

 

The clastogenicity of sodium naphthenate was assessed in anin vitrochromosome aberration test, conducted according to the NTP test protocol (equivalent to OECD Test Guideline 473). CHO cells were treated with the test material at up to 250 μg/mL both in the presence and absence of S9, alongside appropriate vehicle and positive controls. No evidence of clastogenicity was observed (National Toxicology Program, 1993c). In a similar non-guideline investigation, calcium naphthenate (in an unspecified ‘carrier oil’) did not produce chromosome aberrations in rat liver RL4 cells when tested at up to 250 μg/mL in the absence of S9 (Shell Research Ltd, 1983).

 

In an in vitro sister chromatid exchange (SCE) assay with CHO cells, sodium naphthenate was tested in the absence of S9 at concentrations of 17-500 μg/mL (Trial 1) and 100-250 μg/mL (Trial 2) and in the presence of S9 at 17-500 μg/mL. Sodium naphthenate was negative with S9 but weakly positive to positive without S9 (Trials 1 and 2) (National Toxicology Program, 1993c). Although a positive result was obtained in 2 separate runs without S9, the validity of these results is questionable since the occurrence of cytotoxicity was not well documented.

Justification for classification or non-classification

Not classified for genetic toxicity. Negative results in all studies conducted.