Potassium isooctadecanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Amest Test: Potassium isostearate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

MLA: Potassium isostearate, tested up to the cytotoxic concentration of 100 µg/mL medium, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. In addition, no test item-related change was noted in the ratio of small to large mutant colonies. Therefore the test item also did not exhibit any clastogenic potential at the concentration-range investigated.

MNT: Under the present test conditions, potassium isostearate tested up to the cytotoxic concentration of 100 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) using human peripheral lymphocytes revealed no indications of chromosomal damage in the in vitro micronucleus test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Product description: Potassium isooctadecanoate/potassium isostearate
Name: Isooctadecanoic acid, potassium salt
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (UVCB)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test). Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment at the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed at the following concentrations:

Experiment I:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment II:
5.0, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate

As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main experiment I.

Vehicle / solvent:

Water; The test item was completely dissolved in aqua ad iniectabilia at the evaluated concentrations.

Untreated negative controls:

yes

Remarks:

Aqua dest.

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD; 4-nitro-o-phenylene-diamine: TA 98, TA 1537 (without metabolic activation) 2-AA; 2-aminoanthracene: S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA (with metabolic activation)

Details on test system and experimental conditions:

Test System

Tester strains TA 98, TA 1535 and E. coli were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland. All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth). The tester strains TA 98 and TA 100 contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms. The tester strain E. coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed.

Mammalian Microsomal Fraction S9 Mix

The bacteria most commonly used in these reverse mutation assays do not possess the enzyme system which, in mammals, is known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in the form of mammalian microsome enzyme activation mixture.

S9 Homogenate

The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations are performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ≤-75 °C.

The protein concentration in the S9 preparation (Lot: 280716) was 30.3 mg/mL. In Lot 190416 the protein concentration was 34.4 mg/mL and was adjusted to 30 mg/mL.

Preparation of S9 Mix
The S9 mix preparation was performed according to Ames et al.

Evaluation criteria:

Criteria of Validity

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2013 -2015).
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.
- at least five different concentrations of each tester strain are analysable.

Evaluation of Mutagenicity

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.

Statistics:

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

ethanol

Untreated negative controls validity:

valid

Remarks:

aqua dest.

Positive controls validity:

valid

Remarks:

4-NOPD (-S9) and 2-AA (+S9)

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

ethanol

Untreated negative controls validity:

valid

Remarks:

aqua dest.

Positive controls validity:

valid

Remarks:

NaN3 (-S9) and 2-AA (+S9)

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

ethanol

Untreated negative controls validity:

valid

Remarks:

aqua dest.

Positive controls validity:

valid

Remarks:

NaN3 (-S9) and 2-AA (+S9)

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

ethanol

Untreated negative controls validity:

valid

Remarks:

aqua dest.

Positive controls validity:

valid

Remarks:

NaN3 (-S9) and 2-AA (+S9)

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

ethanol

Untreated negative controls validity:

valid

Remarks:

aqua dest.

Positive controls validity:

valid

Remarks:

MMS (-S9) and 2-AA (+S9)

Remarks on result:

other: Plate-incorporation Test

Results

The test item isooctadecanoic acid, potassium salt was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I and II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment II:

5.0, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate

Precipitation of the test item was observed in all tester strains used in experiment I at concentrations of 2500 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation). In experiment II precipitation of the test item was observed in all tester strains used at concentrations of 1580 µg/plate and higher (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II. The reduction in

the number of revertants down to a mutation factor of ≤ 0.5 found in experiment I in tester strain TA 1537 at concentrations of 2500 and 5000 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of concomitant clearing of the background lawn and the fact that this effect was not reproducible in experiment II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with isooctadecanoic acid, potassium salt at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Isooctadecanoic acid, potassium salt did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, isooctadecanoic acid, potassium salt is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In an OECD 471 GLP study isooctadecanoic acid, potassium salt did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Isooctadecanoic acid, potassium salt is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Product description: Potassium isooctadecanoate/potassium isostearate
Name: Isooctadecanoic acid, potassium salt
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (UVCB)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Target gene:

Thymidine kinase (TK) locus in L5178Y TK +/- mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK +/- mouse lymphoma cells

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on the results of the preliminary study five concentrations of 6.25, 12.5, 25, 50 and 100 µg/mL medium for the experiments without and with metabolic activation were employed in the mutagenicity tests.

Vehicle / solvent:

highly purified water; The test item was completely dissolved in aqua ad iniectabilia at the evaluated concentrations.

Untreated negative controls:

yes

Remarks:

highly purified water in culture medium

Negative solvent / vehicle controls:

yes

Remarks:

highly purified water

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

Assay acceptance criteria

An assay is considered acceptable for evaluation of the test results only if all of the criteria given below are satisfied.
The activation and non-activation portions of the mutation assays are usually performed concurrently, but each portion is in fact an independent assay with its own positive and negative controls. The activation or non-activation assays would be repeated independently, as needed, to satisfy the acceptance and evaluation criteria.
- Adequate number of cells and concentrations should be analysable.
- The criteria for the selection of highest concentration are consistent with the guidline.

a) Data of the untreated/solvent control (Mutant Frequency, Cloning Efficiency and the Suspension Growth) meet the IWGT MLA Workgroup acceptance criteria (M.M. Moore et al. 2006). Relevant data is given below:
Parameter MLA (Microwell Method)
Mutant Frequency (MF) 50 - 170 x 10^-6
Cloning Efficiency (CE) 65 - 120 %
Suspension Growth (SG) 8 - 32 fold (3-4 hour treatment), 32-180 fold (24-hour treatment)

b) The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF, or the positive control has an increase in the small colony MF of at least 150 x 10^6 above that seen in the concurrent untreated/solvent control (a small colony IMF of 150 x 10^-6).

c) Mutation Frequencies of both, negative and positive controls fall within the normal range (historical data).

Evaluation criteria:

Assay evaluation criteria

For the MLA, significant work on biological relevance and criteria for a positive response has been conducted by The Mouse Lymphoma Expert Workgroup of the IWGT (M.M. Moore et al., 2006). Therefore, the interpretation of test chemical results is based on those recommendations:
To define positive and negative results and to assure that the increased MF is biologically relevant instead of a statistical analysis (generally used for other tests), the interpretation relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated as the Global Evaluation Factor (GEF). The GEF (126 x 10-6) is based on the analysis of the distribution of the negative control MF data from participating laboratories (M.M. Moore et al., 2006). Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

highly purified water

Untreated negative controls validity:

valid

Remarks:

highly purified water

Positive controls validity:

valid

Remarks:

3-Methylcholanthrene (+S9), Methyl methanesulfonate (-S9)

Additional information on results:

No changes in pH or osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium.

Concentration selection

A preliminary cytotoxicity study was performed to establish an appropriate concentration range for the mutation experiment. This study was performed without and with S9 metabolic activation.

A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000 and 2000 µg Potassium isostearate/mL medium were tested for cytotoxicity.

Based on the results of the preliminary study five concentrations of 6.25, 12.5, 25, 50 and 100 µg/mL medium for the experiments without and with metabolic activation were employed in the mutagenicity tests.

The separation factor of 2 was used. No increase in the mutant frequency was observed. Hence, it was considered acceptable not to add any further lower concentrations, as these additional lower concentrations would provide no further information.

Results

A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation. A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000 and 2000 µg Potassium isostearate/mL medium were tested for cytotoxicity. Complete cytotoxicity (no survival) was noted starting at a concentration of 100 µg/mL in the absence and presence of metabolic activation (24-hour or 3-hour exposure, respectively). Test item precipitation was noted macroscopically at concentrations of 1000 and 2000 µg/mL medium in both experiments. Hence, 100 µg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation. Methyl methanesulfonate (13 or 12 µg/mL) was employed as a positive control in the absence of exogenous metabolic activation and 3 Methylcholanthrene (1.0 µg/mL) in the presence of exogenous metabolic activation. In the main study, cytotoxicity (decreased relative total growth) was noted at concentrations of 50 and 100 µg Potassium isostearate/mL medium in all experiments without and with metabolic activation. In addition, cytotoxicity (decreased relative total growth) was noted at a concentration of 25 µg/mL medium in the second experiment with S9 mix. The negative controls had mutation frequencies of 158.39 or 92.54 mutant colonies per 10^6 cells in the experiments without metabolic activation (3- or 24 hour exposure, respectively) and 167.70 or 55.13 mutant colonies per 10^6 cells in the experiment with metabolic activation and, hence, were all well within the historical data-range.The mutation frequencies of the cultures treated with Potassium isostearate ranged from 60.50 to 155.53 mutant colonies per 10^6 cells (3 hour exposure), from 122.11 to 143.75 mutant colonies per 10^6 cells (24-hour exposure) in the experiments without metabolic activation. The highest concentration of 100 µg/mL medium was almost completely or complete cytotoxic (RTG=2 or 0 in the first or second assay, respectively) and, hence not used for evaluation for mutant colonies. In the experiments with metabolic activation, mutation frequencies ranged from 142.21 to 165.12 mutant colonies per 10^6 cells (3-hour exposure, first assay) and from 58.55 to 115.22 mutant colonies per 106 cells (3-hour exposure, second assay). The highest concentration of 100 µg/mL medium was almost completely or complete cytotoxic (RTG=2 or 0 in the first or second assay, respectively) and, hence not used for evaluation for mutant colonies. The results were within the range of the negative control values and the normal range of 50 to 170 mutants per 10^6 viable cells and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

In addition, no increase in the ratio of small to large mutant colonies, ranging from 0.19 to 1.07 for Potassium isostearate-treated cells and ratios of 0.26 and 0.56 for the negative controls was observed.

The positive controls Methyl methanesulfonate (MMS) and 3-Methylcholanthrene (3 MC) caused pronounced increases in the mutation frequency of 774.63 and 730.89 mutant colonies per 10^6 cells in the case of MMS and of 876.76 and 930.07 mutant colonies per 10^6 cells in the case of 3-MC. As the increase in the small colony mutation frequency was at least 150 x 10^-6 above the concurrent negative control, an absolute increase in total mutation frequency was at least 300 x 10^-6 for the positive controls and the mean relative total growth (RTG) greater than or equal to 10%, the acceptance criteria for the positive controls were met.

The calculations of suspension growth of the negative controls were in the acceptance criteria range between 8 and 32 following 3-hour treatments or between 32 and 180 following 24-hour treatments. The mean cloning efficiencies (CE = PE x 100) of the negative controls from the Mutation Experiments were between the range 65% to 120%. Hence, the acceptance criteria were met.

Plating efficiency and cell growth rate - without S9 activation

1st experiment - 3 hour exposure

Culture number	Concentration of test item [µg/ml]	Wells containing cells of 192 wells	Empty wells of toal 192 wells	P	PE (viable cells)	RPE % (viable cells)	SG	RSG %	RTG
1	0 (control)	130	62	1.1304	0.7065	100	29.4	100	100
6	6.25	128	64	1.0986	0.6866	97	29.8	101	99
5	12.5	134	58	1.1971	0.7482	106	26.8	91	97
4	25	126	66	1.0678	0.6674	94	31.0	106	100
3	50	104	88	0.7802	0.4876	69	7.5	26	18
2	100	20	172	0.1100	0.0688	10	5.1	17	2
8	MMS 13	85	107	0.5847	0.3654	52	18.5	63	33

Plating efficiency and cell growth rate - without S9 activation

2nd experiment - 24 hour exposure

Culture number	Concentration of test item [µg/ml]	Wells containing cells of 192 wells	Empty wells of toal 192 wells	P	PE (viable cells)	RPE % (viable cells)	SG	RSG %	RTG
1	0 (control)	133	59	1.180	0.7375	100	50.2	100	100
6	6.25	125	67	1.0528	0.6580	89	66 .5	132	118
5	12.5	130	62	1.1304	0.7065	96	70.5	140	134
4	25	127	65	1.0831	0.6769	92	61.8	123	113
3	50	66	126	0.4212	0.2633	36	27.4	54	19
2	100	0	192	0.0000	0.0000	0	0.0	0	0
8	MMS 12	63	129	0.3977	0.2486	34	17.4	35	12

Plating efficiency and cell growth rate - with S9 activation

1st experiment - 3 hour exposure

Culture number	Concentration of test item [µg/ml]	Wells containing cells of 192 wells	Empty wells of toal 192 wells	P	PE (viable cells)	RPE % (viable cells)	SG	RSG %	RTG
9	0 (control)	129	63	1.144	0.6965	100	26.4	100	100
14	6.25	125	67	1.0528	0.6580	94	29.8	113	107
13	12.5	125	67	1.0528	0.6580	94	27.9	105	100
12	25	129	63	1.1144	0.6965	100	30.7	116	116
11	50	53	139	0.3230	0.2019	29	18.3	69	20
10	100	21	171	0.1158	0.0724	10	6.3	24	2
16	3-MC 1.0	66	126	0.4212	0.2633	38	15.9	60	23

Plating efficiency and cell growth rate - with S9 activation

2nd experiment - 3 hour exposure

Cullture number	Concentration of test item [µg/ml]	Wells containing cells of 192 wells	Empty wells of toal 192 wells	P	PE (viable cells)	RPE % (viable cells)	SG	RSG %	RTG
9	0 (control)	128	64	1.0986	0.6866	100	19.6	100	100
14	6.25	140	52	1.3063	0.8164	119	11.6	59	71
13	12.5	125	67	1.0528	0.6580	96	18.1	92	88
12	25	85	107	0.5847	0.3654	53	18.0	92	49
11	50	65	127	0.4133	0.2583	38	5.5	28	11
10	100	0	192	0.0000	0.0000	0	6.7	34	0
16	3-MC 1.0	62	130	0.3900	0.2438	36	7.1	36	13

3-MC = 3-Methylcholanthrene

MMS = Methylmethansulfonate

P = probable number of clones/well

(R) PE = (relative) plating efficiency

RTG = relative total growth

RSG = relative suspension growth

SG = suspension growth

Mutant Frequency/10^6 cells without metabolic activation

1st experiment - 3 hour exposure

Culture number	Concentration of test item [µg/ml]	Wells containing mutants of total 384 wells	Pm	PEm (mutant cells)	PE (viable cells)	CE	MF/10^6	small colony MF/10^6
1	0 (control)	77	0.2238	1.1190E-04	0.7065	70.65	158.39	30.15
6	6.25	64	0.1823	9.1150E-05	0.6866	68.66	132.76	25.05
5	12.5	78	0.2271	1.1355E-04	0.7482	74.82	151.76	39.43
4	25	72	0.2076	1.0380E-04	0.6674	66.74	155.53	40.08
3	50	22	0.0590	2.9500E-05	0.4876	48.76	60.50	10.77
2	100	2	-	-	-	-	-	-
8	MMS 13	166	0.5661	2.8305E-04	0.3654	36.54	774.63	388.89

Mutant Frequency/10^6 cells without metabolic activation

2nd experiment - 24 hour exposure

Culture number	Concentration of test item [µg/ml]	Wells containing mutants of total 384 wells	Pm	PEm (mutant cells)	PE (viable cells)	CE	MF/10^6	small colony MF/10^6
1	0 (control)	49	0.1365	6.8259E-05	0.7375	73.75	92.54	26.98
6	6.25	57	0.1607	8.0350E-05	0.6580	65.80	122.11	42.71
5	12.5	61	0.1730	8.6500E-05	0.7065	70.65	122.43	43.74
4	25	62	0.1761	8.8050E-05	0.6769	67.69	130.08	64.26
3	50	28	0.0757	3.7850E-05	0.2633	26.33	143.75	55.26
2	100	0	-	-	-	-	-	-
8	MMS 12	117	0.3634	1.8170E-04	0.2486	24.86	730.89	450.12

Mutant Frequency/10^6 cells with metabolic activation

1st experiment - 3 hour exposure

Culture number	Concentration of test item [µg/ml]	Wells containing mutants of total 384 wells	Pm	PEm (mutant cells)	PE (viable cells)	CE	MF/10^6	small colony MF/10^6
9	0 (control)	80	0.2336	1.1680E-04	0.6965	69.65	167.70	44.36
14	6.25	70	0.2012	1.0060E-04	0.6580	65.80	152.89	44.83
13	12.5	75	0.2173	1.0865E-04	0.6580	65.80	165.12	24.09
12	25	69	0.1981	9.9050E-05	0.6965	69.65	142.21	34.46
11	50	23	0.0681	3.0900E-05	0.2019	20.19	153.05	32.44
10	100	4	-	-	-	-	-	-
816	3-MC 1.0	142	0.4617	2.3085E-04	0.2633	26.33	876.76	418.72

Mutant Frequency/10^6 cells with metabolic activation

2nd experiment - 3 hour exposure

Culture number	Concentration of test item [µg/ml]	Wells containing mutants of total 384 wells	Pm	PEm (mutant cells)	PE (viable cells)	CE	MF/10^6	small colony MF/10^6
9	0 (control)	28	0.0757	3.7850E-05	0.6866	68.66	55.13	19.23
14	6.25	35	0.0956	4.7800E-05	0.8164	81.64	58.55	22.72
13	12.5	34	0.0927	4.6350E-05	0.6580	65.80	70.44	28.19
12	25	31	0.0842	4.2100E-05	0.3654	36.54	115.22	39.82
11	50	12	0.0317	1.5850E-05	0.2583	25.83	61.36	25.36
10	100	0	-	-	-	-	-	-
16	3-MC 1.0	140	0.4535	2.2675E-04	0.2438	24.38	930.07	506.36

MMS = Methylmethansulfonate

3-MC = 3 -Methylcholanthrene

Pm = probable number of clones/well

PE = plating efficiency

CE = cloning efficiency

MF/10^6 = mutant frequency per 10^6 cells   

Conclusions:

Under the present test conditions, potassium isostearate, tested up to the cytotoxic concentration of 100 µg/mL medium, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. In addition, no test item-related change was noted in the ratio of small to large mutant colonies. Therefore, potassium isostearate also did not exhibit any clastogenic potential at the concentration-range investigated.

Executive summary:

In a Klimisch 1 OECD 490 GLP study, potassium isostearate, tested up to the cytotoxic concentration of 100 µg/mL medium, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions, the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions. In addition, no test item-related change was noted in the ratio of small to large mutant colonies. Therefore, potassium isostearate also did not exhibit any clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that potassium isostearate, tested up to the cytotoxic concentration of 100 µg/mL medium, neither induced mutations nor had any chromosomal aberration potential.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Product description: Potassium isooctadecanoate/potassium isostearate
Name: Isooctadecanoic acid, potassium salt
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (UVCB)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human peripheral blood was obtained by venipuncture from young, healthy, non-smoking male individuals (31 to 38 years of age) with no known recent exposures to genotoxic chemicals or radiation, and collected in heparinised vessels. Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin . The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

CytoB (Cytochalasin B)
The concentration used for this assay was 5 µg/mL.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg potassium isostearate/mL medium were employed. Complete cytotoxicity (no survival) was noted starting at a concentration of 100 µg/mL in the absence and presence of metabolic activation (24-hour or 4-hour exposure, respectively).
Hence, 100 µg/mL and one additional concentration of 200 µg/mL medium for the 4-hour exposure experiments were employed as the top concentrations for the genotoxicity tests without and with metabolic activation.

Vehicle / solvent:

highly purified water; The test item was completely dissolved in aqua ad iniectabilia at the evaluated concentrations.

Untreated negative controls:

yes

Remarks:

highly purified water

Negative solvent / vehicle controls:

yes

Remarks:

highly purified water

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine

Details on test system and experimental conditions:

Culture establishment

Human peripheral blood was obtained by venipuncture from young, healthy, non-smoking male individuals (31 to 38 years of age) with no known recent exposures to genotoxic chemicals or radiation, and collected in heparinised vessels. Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of Chromosome complete culture medium with Phytohemagglutinin and 1% Penicillin/Streptomycin . The tubes are sealed and incubated at 37°C, and shaken occasionally to prevent clumping.

Evaluation criteria:

The assay demonstrates its ability to reliably and accurately detect substances of known aneugenic and clastogenic activity, with and without metabolic activation.

Acceptance of a test is based on the following criteria:

- The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database (Poisson-based 95% control limits). Where concurrent negative control data fall outside the 95% control limits, they may be acceptable for inclusion in the historical control data as long these data are not extreme outliers.
- Concurrent positive controls induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
- Adequate number of cells, cell proliferation criteria and concentrations are analysable.

Vehicle control and untreated cultures give reproducibly low and consistent micronucleus frequencies. Data from vehicle and positive controls are used to establish historical control ranges. These values are used in deciding the adequacy of the concurrent vehicle controls or positive controls for an experiment.

Statistics:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)

When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:

- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).

The test chemical is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Key result

Species / strain:

lymphocytes: Human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

highly purified water

Untreated negative controls validity:

valid

Remarks:

highly purified water

Positive controls validity:

valid

Remarks:

Colchicine and Mitomycin C in aqua ad iniectabilia (-S9); Cyclophosphamide in aqua ad iniectabilia (+S9);

Additional information on results:

No changes in pH or osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium.

Results

In the main study cytotoxicity was noted at the top concentration of 100 µg Potassium isostearate/mL medium in the experiments without and with metabolic activation. 200 µg potassium isostearate/ml medium were completely cytotoxic.

Mitomycin C (at 0.2 µg/mL) and colchicine (at 0.02 µg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 µg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of 12.5, 25, 50 and 100 or 6.25, 12.5, 25, 50 and 100 µg Potassium isostearate/mL medium in the absence of metabolic activation (4- and 24-hour exposure, respectively) ranged from 4.0 to 12.0 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 100 µg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test frequencies of 4.0 or 8.0 micronucleated cells per 1000 binucleated cells for the 4-hour and 24-hour exposure, respectively, were observed. The vehicle result was within the historical control ranges. In the positive control cultures the micronucleus frequencies were increased to 21.0 or 23.0 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of 12.5, 25, 50 and 100 µg Potassium isostearate/mL medium (4-h exposure) in the presence of metabolic activation ranged from 5.5 to 9.0 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 100 µg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a mean frequency of 7.0 micronucleated cells per 1000 binucleated cells was observed. The vehicle result was within the historical control ranges. In the positive control culture the micronucleus frequency was increased to 27.0 micronucleated cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage. A summary of the results (means of the two replicate cultures) of this study is listed in Tables below. No changes in pH or in osmolality of the test item formulations compared to the negative control were noted up to the top concentration of 2000 µg/mL medium (preliminary test).

Experiment without metabolic activation (S9 mix) - 4 -h exposure

Culture number	Concentration [µl/mk medium]	mononucleate	Number of binucleate cells#	multinucleate	CBPI	RI [%]	Number of binucleate cells scored	Number of multinucleated cells per 1000 binucleate cells	Significance chi2-test
1	0 (vehicle)	265	236	8	1.50	100	1000	6	-
9	0 (vehicle)	228	256	16	1.58	100	1000	2	-
6	12.5	167	294	39	1.74	148	1000	3	n.s.
14	12.5	157	307	36	1.76	131	1000	5	n.s.
5	25	206	260	34	1.66	132	1000	6	n.s.
13	25	192	284	24	1.66	114	1000	7	n.s.
4	50	278	211	11	1.47	94	1000	7	n.s.
12	50	217	270	13	1.59	102	1000	6	n.s.
3	100	379	101	20	1.28	56	1000	5	n.s.
11	100	403	83	14	1.22	38	1000	4	n.s.
2 & 10	200	no binucleated cells could be evaluated due to cytotoxicity of the test item	-	-	-	-	-	-	-
7 - Mitomycin C	0.2	242	252	6	1.53	106	1000	22	s.
15 -  Mitomycin C	0.2	277	220	3	1.45	78	1000	20	s.

Experiment without metabolic activation (S9 mix) - 24 -h exposure

Culture number	Concentration [µl/mk medium]	mononucleate	Number of binucleate cells#	multinucleate	CBPI	RI [%]	Number of binucleate cells scored	Number of multinucleated cells per 1000 binucleate cells	Significance chi2-test
1	0 (vehicle)	278	207	15	1.47	100	1000	8	-
9	0 (vehicle)	283	203	14	1.46	100	1000	8	-
6	6.25	276	214	10	1.47	100	1000	16	n.s.
14	6.25	293	200	7	1.43	93	1000	8	n.s.
5	12.5	274	213	13	1.48	102	1000	11	n.s.
13	12.5	295	194	11	1.43	93	1000	10	n.s.
4	25	300	194	6	1.41	87	1000	5	n.s.
12	25	287	203	10	1.45	98	1000	3	n.s.
3	50	286	204	10	1.45	96	1000	3	n.s.
11	50	289	203	8	1.44	96	1000	10	n.s.
2	100	386	102	12	1.25	53	1000	10	n.s
10	100	399	94	7	1.22	38	1000	10	n.s.
7 -  Colchicine	0.02	333	156	11	1.36	77	1000	27	s.
15- Colchicine	0.02	232	254	14	1.56	122	1000	19	s.

Experiment with metabolic activation (S9 mix) - 4 -h exposure

Culture number	Concentration [µl/mk medium]	mononucleate	Number of binucleate cells#	multinucleate	CBPI	RI [%]	Number of binucleate cells scored	Number of multinucleated cells per 1000 binucleate cells	Significance chi2-test
1	0 (vehicle)	247	226	27	1.56	100	1000	6	-
9	0 (vehicle)	215	246	39	1.65	100	1000	8	-
6	12.5	248	213	39	1.58	104	1000	8	n.s.
14	12.5	279	202	19	1.48	74	1000	7	n.s.
5	25	223	255	22	1.60	107	1000	8	n.s.
13	25	195	265	40	1.69	106	1000	10	n.s.
4	50	276	205	19	1.49	88	1000	9	n.s.
12	50	292	194	14	1.44	68	1000	6	n.s.
3	100	348	144	8	1.32	57	1000	4	n.s.
11	100	402	96	2	1.20	31	1000	7	n.s.
2	200	no binucleated cells could be evaluated due to cytotoxicity of the test item	-	-	-	-	-	-	n.s
10	200	no binucleated cells could be evaluated due to cytotoxicity of the test item	-	-	-	-	-	-	n.s.
7 -  Cyclophosphamide	20	325	169	6	1.36	64	1000	24	s.
15 - Cyclophosphamide	20	322	173	5	1.37	57	1000	30	s.

n.s. = not significantly different from negative control

s. = significantly different from negative control

# = at least a total of 500 cells have to be scored

CBPI = Cytokinesis block proliferation index

RI = replicative index

Conclusions:

Under the present test conditions, potassium isostearate tested up to the cytotoxic concentration of 100 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.

Executive summary:

In a Klimisch 1 OECD 487 GLP study, potassium isostearate tested up to the cytotoxic concentration of 100 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test. The results for the vehicle controls were within historical control range. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available