Fatty acids, tallow, calcium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro: - gene mutation (bacterial reversion assay/Ames test): Negative - gene mutation (mammalian cell TK+/- assay in L5178Y mouse lymphoma cells): Negative - cytogenicity (chromosomal aberration assay in human lymphocytes): Negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated 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.

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

Migrated to IUCLID6: 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

Migrated to IUCLID6: 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

Migrated to IUCLID6: 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

Migrated to IUCLID6: 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

Migrated to IUCLID6: 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

Migrated to IUCLID6: 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, eg 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 109 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

Remarks:

Migrated from field 'Test system'.

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 (migrated information):
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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The substances in the category are considered to be similar on the basis that they have common structures of a calcium ion varying only by the length of the fatty acid chain and the presence of unsaturated and/or hydroxyl functional groups. As a result it is expected that the substances will have similar, predictable properties. REACH Annex V, Entry 9, groups fatty acids and their potassium, sodium, calcium and magnesium salts, including C6 to C24, predominantly even-numbered, unbranched, saturated or unsaturated aliphatic monocarboxylic acids. Provided that they are obtained from natural sources and are not chemically modified, the substances included in REACH Annex V, Entry 9 are exempt from registration, unless they are classified as dangerous (except for flammability, skin irritation or eye irritation) or they meet the criteria for PBT/vPvB substances. The metal fatty acid substances in the category are therefore not expected to be hazardous. Due to the close structural similarity and the narrow range of carbon chain numbers covered in this category, the genetic toxicity properties are expected to be predictable across the category.

Since REACH Annex V groups together calcium, potassium, sodium and magnesium salts of C6 to C24 fatty acids as being potentially exempt from registration, these metal cations are therefore not considered to contribute to any health hazard. On this basis, relevant published or proprietary data on any potassium, sodium or magnesium salt within the fatty acid category range of C14 to C22 can be used to read across to the calcium salts of C14-C22 fatty acid category.

Lithium salts of fatty acids are not included in REACH Annex V as being potentially exempt from registration. For these salts it is expected that the lithium cation would be the species with the potentially higher toxicity profile when compared to calcium, potassium, sodium, and magnesium cations. However, the substance fatty acids C18 (unsaturated) lithium salts contains a fatty acid anion that falls within the C14-C22 category. Experimental data for the mammalian toxicity Annex VIII endpoints have been generated on this substance and the results obtained are relevant to read across to the calcium salts of C14-C22 fatty acids either in a weight of evidence approach or as key studies due to the structural similarity and its position within the category fatty acid range.

It is relevant to consider metal cations and the fatty acid anions in the review of genotoxic potential of the calcium fatty acid salts. Castor oil (C18 hydroxylated) and magnesium stearate (C18) have been tested in the Ames test with negative results, as reported in the API Robust Summaries (2008, referencing NTP 1992 and Litton Bionetics 1976, respectively).Stearic acid was tested for mutagenicity with and without S9 in the Ames test (reported in CIR 1987) and no mutagenic activity was observed. A sister chromatid exchange study on oleic acid (C18 unsaturated) was reported in the same review, using Chinese hamster V79 lung fibroblasts. The mean number of SCE’s per metaphase was similar to the control, but higher incidences of aneuploidy were apparently seen with the oleic acid cultures. However, oleic (C18 unsaturated) and lauric (C12) acids have been considered as inhibitors of mutagenicity produced by positive control substances such as N-nitrosopyrrolidine and sodium azide (CIR 1987). Additional reporting of fatty acid genetic toxicity is presented in the HERA review (HERA 2002). In this review, it is stated that fatty acids are negative in in vitro bacterial systems used in the Ames test. These included capric acid (C10), lauric acid (C12), stearic acid (C18), oleic acid (C18 unsaturated), and fatty acids C18-22. Each of these substances has been subjected to reversion tests in Escherichia coli or Salmonella typhimurium strains with and without metabolic activation.

Thus there is a significant amount of published information to indicate that fatty acids and their salts are not likely to be mutagenic, particularly in view of the significant normal human intake of fatty acids in the diet.

Key bacterial reversion assays (Ames test) have been conducted on lithium salts of fatty acids within the C14-C22 carbon chain range of the calcium fatty acid salts.The substances were lithium myristate (C14), lithium 12-hydroxystearate (C18) and lithium behenate (C22), together with fatty acids C18 (unsaturated) lithium salts. In addition, a chromosomal aberration assay and a mouse lymphoma assay have been performed on the latter lithium salt. In all cases the results were negative.

Since none of the lithium, and calcium metal cations are considered to be mutagens, the negative experimental data from the lithium fatty acid salts, can be read across to all members of the calcium fatty acid salts category.

Overall, there is no evidence of genotoxicity associated with the substances in the category.

References

American Petroleum Institute (2008) Robust Summary of Information on Grease Thickeners (Creation date: October 11, 2003. Printing date: February 20, 2009. Last update: October 20, 2008. Document date: January 11, 2005)

CIR (Cosmetics Ingredients Review) (1987) Final report on the safety assessment of oleic acid, lauric acid, palmitic acid, myristic acid and stearic acid. Journal of American Toxicologists, vol. 6, iss. 3, pp. 321-401

HERA (Human Health and Environmental Risk Assessment on ingredients of European household cleaning products) (2003) Fatty Acid Salts (Soap) Environmental and Human Health Risk Assessment

National Toxicology Program (1992) Toxicity Studies of Castor Oil in F344/N Rats and B6C3F₁mice (Dosed Feed Studies)

Justification for selection of genetic toxicity endpoint
This substance is a representative fatty acid salt that can be read across to the calcium salts of C14-C22 fatty acids category.

Justification for classification or non-classification

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