Docosanoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies on genotoxicity are available for the following fatty acid category members:
in-vitro:
- Gene mutation in bacteria:

CAS# 112-85-6, C22: Ames (Bacterial reverse mutation assay, OECD 471: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A, with and without metabolic activation, RL1): negative (Nakajima 2002)

CAS# 112-85-6, C22: Ames RL4: negative (Gloxhuber and Wallat 1981)

- Gene mutation in mammalian cells:
CAS# 334-48-5, C10: Mouse Lymphoma Assay in-vitro (Gene mutation in mammalian cells, TK locus, OECD 476, with and without metabolic activation, RL2): negative (Trenz 2010)

CAS# 60-33-3, C 18:2: Mouse Lymphoma Assay in-vitro RL2: negative (Seifried et al., 2006)

CAS# 463-40-1, C 18:3: Mouse Lymphoma Assay in-vitro RL2: negative (Seifried et al., 2006)

- Cytogenicity in mammalian cells:
CAS# 112-85-6, C22: Chromosomal Aberration test in-vitro (OECD 473, with and without meatbolic activation, RL1): negative (Nakajima 2002)

in-vivo:
No data available.
All available data on genotoxicity showed that fatty acids are not genotoxic.

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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP - Guideline study, tested with the source substance CAS 112-85-6. In accordance to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium strains), trp operon (E. coli strain)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from livers of male Sprague-Dawley rats

Test concentrations with justification for top dose:

156, 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 - 0.1 µg/plate); sodium azide (0.5 µg/pl.); 9-aminoacridine hydrochloride (80 µg/pl.); +S9: 2-aminoanthracene (0.5 - 10 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

An increase twice the number of revertant colonies in test concentration plates compared to the solvent control plates and an observable dose-dependency is considered as a positive result.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Visible precipitation occurred at the end of the exposure period at all test concentrations.

Table 1: Test Results of Experiment 1 

EXPERIMENT 1 (Preincubation Test)
S9-Mix	Without
Test item (µg/plate)	TA 100	TA 1535	E. coli WP2 uvrA	TA 98	TA 1537
DMSO	95 ± 2	15 ± 1	25 ± 2	23 ± 3	9 ± 2
156*	94 ± 3	11 ± 1	27 ± 3	21 ± 2	8 ±3
313*	94 ± 4	12 ± 2	24 ± 1	21 ± 4	10 ± 2
625*	89 ± 2	14 ± 3	26 ± 1	18 ± 2	8 ± 1
1250*	82 ± 4	13 ± 1	25 ± 3	21 ± 3	8 ± 3
2500*	80 ± 2	11 ± 1	26 ± 1	17 ± 3	10 ± 4
5000*	78 ± 2	9 ± 2	28 ± 1	14 ± 4	10 ± 3
AF-2	687 ± 12	-	173 ± 10	460 ± 27	-
NaN3	-	422 ± 7	-	-	-
ACR	-	-	-	-	491 ± 28
S9-Mix	With
Test item (µg/plate)	TA 100	TA 1535	E. coli WP2 uvrA	TA 98	TA 1537
DMSO	112 ± 4	13 ± 1	31 ± 2	33 ± 2	7 ± 1
156*	103 ± 4	12 ± 3	27 ± 3	30 ± 3	10 ± 3
313*	106 ± 10	10 ± 4	26 ± 1	34 ± 4	11 ± 3
625*	97 ± 3	12 ± 2	27 ± 1	29 ± 4	10 ± 3
1250*	97 ± 5	12 ± 4	28 ± 5	30 ± 2	10 ± 1
2500*	98 ± 3	12 ± 3	27 ± 3	30 ± 2	10 ± 3
5000*	99 ± 9	9 ± 2	28 ± 2	29 ± 1	9 ± 1
2-AA	723 ± 12	383 ± 5	516 ± 5	366 ± 19	172 ± 8

* precipitation occurred at the end of the exposure

AF-2: 2-(2 furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/plate for TA 100 and E. coli WP2 uvrA; 0.1 µg/plate for TA 98)

NaN3: sodium azide (0.5 µg/plate)

ACR: 9-aminoacridine hydrochloride (80 µg/plate)

2-AA: 2-aminoanthracene (1 µg/plate for TA 100; 2 µg/plate for TA 1535 and TA 1537; 10 µg/plate for E. coli WP2 uvrA; 0.5 µg/plate for TA 98)

 

 

Table 2: Test Results of Experiment 2 

EXPERIMENT 2 (Preincubation Test)
S9-Mix	Without
Test item (µg/plate)	TA 100	TA 1535	E. coli WP2 uvrA	TA 98	TA 1537
DMSO	95 ± 2	16 ± 2	26 ± 3	23 ± 2	5 ± 1
156*	94 ± 3	14 ± 1	25 ± 1	23 ± 3	6 ± 2
313*	95 ± 3	14 ± 3	26 ± 2	23 ± 4	6 ± 2
625*	90 ± 0	17 ± 1	24 ± 3	22 ± 2	7 ± 0
1250*	89 ± 5	15 ± 1	25 ± 3	21 ± 2	5 ± 2
2500*	85 ± 5	15 ± 2	23 ± 1	20 ± 1	7 ± 1
5000*	88 ± 3	15 ± 3	22 ± 2	20 ± 1	4 ± 1
AF-2	734 ± 14	-	128 ± 6	439 ± 7	-
NaN3	-	433 ± 3	-	-	-
ACR	-	-	-	-	503 ± 5
S9-Mix	With
Test item (µg/plate)	TA 100	TA 1535	E. coli WP2 uvrA	TA 98	TA 1537
DMSO	107 ± 3	15 ± 2	29 ± 2	26 ± 2	14 ± 1
156*	100 ± 2	15 ± 1	28 ± 1	29 ± 2	14 ± 2
313*	99 ± 4	13 ± 1	29 ± 3	30 ± 8	14 ± 1
625*	103 ± 3	14 ± 2	27 ± 2	30 ± 4	15 ± 2
1250*	101 ± 4	12 ± 2	28 ± 5	28 ± 2	13 ± 1
2500*	98 ± 2	13 ± 2	26 ± 2	26 ± 1	11 ± 1
5000*	96 ± 5	10 ± 1	26 ± 2	24 ± 1	12 ± 2
2-AA	678 ± 17	393 ± 5	521 ± 22	423 ± 31	173 ± 13

* precipitation occurred at the end of the exposure

AF-2: 2-(2 furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 µg/plate for TA 100 and E. coli WP2 uvrA; 0.1 µg/plate for TA 98)

NaN3: sodium azide (0.5 µg/plate)

ACR: 9-aminoacridine hydrochloride (80 µg/plate)

2-AA: 2-aminoanthracene (1 µg/plate for TA 100; 2 µg/plate for TA 1535 and TA 1537; 10 µg/plate for E. coli WP2 uvrA; 0.5 µg/plate for TA 98)

Conclusions:

The test substance did not induce gene mutations in the S. typhimurium and E. coli strains. No toxicity was observed up to a concentration of 5000 µg/plate, with or without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung (CHL) cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle-MEM liquid medium
- Properly maintained: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
+S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
-S9 mix (24 h continuous exposure): 0, 350, 700, 1400, 2800 µg/mL
-S9 mix (48-hour continuous exposure): 0, 288, 575, 1150, 2300 µg/mL

Vehicle / solvent:

1.0% carboxymethylcellulose sodium

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: continuous exposure: mitomycin C (0.05 µg/mL for 24 hours and 0.025 µg/mL for 48 hours); short-term exposure: cyclophosphamide (12.5 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration: 6 (short-term exposure), 24, 48 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The frequency of polyploid cells or cells with abnormal structure of each test group were determined according to the criteria of Ishidate.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung (CHL) cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble in water, soluble in alcohol, ether, chloroform and acetone
- Precipitation: observed on the slide of continuous exposure high dose group

RANGE-FINDING/SCREENING STUDIES: see 'Any other information on material and method incl. tables'

COMPARISON WITH HISTORICAL CONTROL DATA: this test was valid, since the frequency of chromosomal aberration in positive control was within background data.

Table 1: Results of growth inhibition test

Test item	Concentration in µg/mL	Survival in %
Exposure period 24 h, without S9 mix
1% CMC▪Na		100
Test substance	272	97.7
	454	95.2
	756	96.5
	1260	83.1
	2100	62.1
	3500	37.8
Exposure period 48 h, without S9 mix
1% CMC▪Na		100
Test substance	272	101.1
	454	104.6
	756	98.9
	1260	89.7
	2100	75.9
	3500	1.3
Exposure period 6 h, without S9 mix
1% CMC▪Na		100
Test substance	272	109.6
	454	93.7
	756	102.5
	1260	110.7
	2100	89.8
	3500	95.1
Exposure period 6 h, with S9 mix
1% CMC▪Na		100
Test substance	272	84.3
	454	85.9
	756	85.3
	1260	86.5
	2100	68.3
	3500	76.3

 

 

Table 2: Results of chromosome aberration test

Test item	Concentration	Aberrant cells in %			Polyploid cells in %
	in µg/mL	with gaps	without gaps
Exposure period 24 h, without S9 mix
1% CMC▪Na		0.5	0.5	0.0
MMC	0.05	56.0	52.5	0.5
Test substance	350	1.0	1.0	0.5
	700	1.0	0.5	0.5
	1400	0.5	0.0	0.0
	2800	Toxic
Exposure period 48 h, without S9 mix
1% CMC▪Na		0.0	0.0	0.5
MMC	0.025	58.5	54.5	0.0
Test substance	288	1.0	1.0	0.0
	575	0.5	0.0	0.5
	1150	2.0	1.5	0.0
	2300	Toxic
Exposure period 6 h, without S9 mix
1% CMC▪Na		1.5	0.5	0.5
CP	12.5	0.5	0.0	0.5
Test substance	875	0.5	0.5	0.0
	1750	3.0	2.5	0.0
	3500	1.0	0.5	0.5
Exposure period 6 h, with S9 mix
1% CMC▪Na		1.5	1.0	0.0
CP	12.5	63.5	61.5	0.0
Test substance	875	2.5	1.5	1.0
	1750	2.0	2.0	0.0
	3500	2.0	2.0	0.0

CMC▪Na: carboxymethylcellulose sodium (solvent)

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

 

Conclusions:

The test substance did not induce structural chromosomal aberrations in the absence or presence of an exogenous metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

28 Jun - 23 Aug 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP- Guideline study (OECD) tested with the source substance CAS 334-48-5. In accordance to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 complete medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital (80 mg/kg bw) and beta-naphtoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

Pre-Test:
Experiment 1:
- with and without metabolic activation: 0.5, 2, 4, 6, 8, 10 mM
Experiment 2:
- without metabolic activation. 0.005, 0.05, 0.2, 0.7, 1.3, 2.0 mM

Main Test:
Experiment 1:
- with metabolic activation: 0.70, 0.82, 0.94, 1.06, 1.18, 1.30, 1.42, 1.54 mM
- without metabolic activation. 0.22, 0.46, 0.58, 0.70, 0.82, 0.94, 1.06, 1.18 mM
Experiment 2:
- with metabolic activation: 1.0, 1.12, 1.24, 1.36, 1.48, 1.60, 1.72, 1.84 mM
- without metabolic activation. 0.0005, 0.001, 0.002, 0.005, 0.01, 0.06, 0.18, 0.3 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI cell culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

since medium was used as solvent, no further solvent control was necessary

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: methylmethanesulfonate (10 µg/mL, dissolved in 0.9% NaCl); ethylemethanesulphanate (200 and 500 µg/mL, dissolved in medium); +S9: benzo(a)pyrene (3.5 µg/mL, dissolved in DMSO (1% final concentration in medium))

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 h (short-term exposure) with and without metabolic activation
Experiment 2: 4 h (short-term exposure) with metabolic activation and 24 h (long-term exposure) without metabolic activation
- Expression time (cells in growth medium): 3 days (short-term exposure) or 2 days (long-term exposure)
- Selection time (if incubation with a selection agent): 11 - 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13 - 18 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; cloning efficiency; mitotic index

OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations

Evaluation criteria:

There are several criteria for a positive result:
- clear and dose-related increase in the mutant frequency
- biologically relevant response (at least a 2-fold increase of mutant frequencies related to the respective negative control values and higher than the historical range of negative controls) for at least one of the dose groups
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/Small colonies ratio (1.5 times the ratio of clastogenic control MMS and/or B[a]P) is an indication for potential clastogenic effects and/or chromosomal aberrations.

The test substance is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at any dose level.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1.54 mM with metabolic activation and at 1.18 mM without metabolic activation in experiment 1, respectively; at 1.84 mM with metabolic activation and at 0.30 mM without metabolic activataion in experiment 2, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: within the physiological range
- Effects of osmolality: within the physiological range
- Precipitation: in the pre-test with metabolic activation from concentrations of 4 mM and higher

RANGE-FINDING/SCREENING STUDIES:
All mutant values were found to be within the range of the historical control data of the test facility BSL Bioservice (about 51 to 170 mutants per 10^6 cells)

COMPARISON WITH HISTORICAL CONTROL DATA:
All mutant values were found to be within the range of the historical control data of the test facility BSL Bioservice (about 51 to 170 mutants per 10^6 cells)

Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. A mutation frequency above 2 in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone), indicated by a low large/small colony ratio (ratio of the clastogenic controls MMS and/or B[a]P with a coefficient of 1.5), is an indication for potential clastogenic effects and/or chromosomal aberrations.

Although in experiment 1 with metabolic activation an increased number of small colonies was noted at doses of 1.30 mM and 1.42 mM (26 and 31 small colonies, respectively, compared to 11 and 9 at control) all dose groups were considered as not clastogenic since no mutagenicity was found at these doses.

All other dose groups in the other experiments were also found not to be clastogenic, respectively.

Table 1: Experiment I - 4 h exposure - With Metabolic Activation

Concentration [mM]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor	Colony Sizing Quotient Large/Small
0	100	100	86.77	1	3.66
0.7	96.63	95.51	95.86	1.10	--
0.82	104.12	92.71	88.56	1.02	--
0.94	109.36	95.60	76.43	0.88	--
1.06	110.11	78.73	78.58	0.91	--
1.18	109.36	60.06	86.55	1.00	--
1.30	116.10	40.14	100.88	1.16	1.77
1.42	112.36	31.61	121.24	1.40	1.55
1.54	96.63	9.84	139.92	1.61	2.78
B[a]P, 3.5 µg/mL	99.63	69.03	623.89	7.19	1.24

B[a]P:Benzo[a]pyrene

  

Table 2: Experiment I - 4 h exposure - Without Metabolic Activation 

Concentration [mM]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor	Colony Sizing Quotient Large/Small
0	100	100	79.39	1	2.75
0.22	100.33	90.09	78.53	0.99	--
0.46	86.38	79.76	124.63	1.57	--
0.58	91.03	79.70	77.88	0.98	--
0.70	98.34	89.53	70.89	0.89	--
0.82	98.34	71.30	69.28	0.87	--
0.94	95.02	64.50	62.74	0.79	1.60
1.06	99.00	47.66	74.59	0.94	3.17
1.18	91.69	11.04	97.49	1.23	1.12
EMS, 500 µg/mL	86.38	62.27	1337.77	16.85	--
MMS, 10 µg/mL	85.71	66.62	841.03	10.59	0.69

EMS:Ethyl methane sulphonate

MMS:Methyl methane sulphonate

 Table 3: Experiment II - 4 h Exposure - With Metabolic Activation

Concentration [mM]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor	Colony Sizing Quotient Large/Small
0	100	100	82.64	1.00	2.07
1	96.97	85.91	82.23	1.00	--
1.12	101.68	89.08	61.54	0.74	--
1.24	98.99	86.20	86.92	1.05	--
1.36	99.66	83.65	75.75	0.92	--
1.48	90.91	64.04	118.05	1.43	--
1.60	96.07	35.51	70.23	0.85	2.38
1.72	91.58	38.25	84.77	1.03	2.13
1.84	98.99	19.98	98.81	1.20	4.73
B[a]P, 3.5 µg/mL	86.20	60.39	829.02	10.03	0.89

B[a]P:Benzo[a]pyrene

 

 

Table 4: Experiment II - 24 h exposure - Without Metabolic Activation

Concentration [mM]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 surviving cells	Mutation factor	Colony Sizing Quotient Large/Small
0	100	100	104.66	1	2.61
0.0005	95.24	94.64	76.34	0.73	--
0.001	101.36	103.58	68.22	0.65	--
0.002	94.56	95.29	66.56	0.64	--
0.005	101.36	105.73	52.63	0.50	--
0.01	102.04	98.98	64.06	0.61	--
0.06	102.72	96.75	61.54	0.59	3.30
0.18	96.60	55.56	91.91	0.88	2.24
0.30	91.16	19.77	99.26	0.95	1.94
EMS, 200 µg/mL	70.75	34.84	2516.55	24.05	--
MMS, 10 µg/mL	59.18	27.33	2625.00	25.08	0.81

EMS: Ethyl methane sulphonate

MMS: Methyl methane sulphonate

Conclusions:

negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria

Genetic toxicity in bacteria was evaluated in a study performed according to GLP and OECD guideline 471and 472, and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals (Nakajima, 2002). S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A were treated with docosanoic acid dissolved in DMSO at concentrations of 156, 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation by S9 mix from livers of male Sprague-Dawley rats induced with phenobarbital and 5,6-benzoflavone. No increase in revertants compared to the vehicle control was observed in any of the strains at all test concentrations. No toxicity was noted up to a concentration of 5000 µg/plate. Thus, docosanoic acid can be regarded as non-mutagenic in bacteria.

The same result was found in another study, where S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were tested with docosanoic acid concentrations of 4, 20, 100, 500, 1250 µg/plate with and without metabolic activation by S9 mix (Gloxhuber and Wallat, 1981). Docosanoic acic did not induce gene mutation in bacteria.

Cytogenicity in mammalian cells

A further in vitro mammalian chromosome aberration test was conducted with docosanoic acid (CAS# 112-85-6) in accordance with GLP and OECD Guideline 473 and Japanese Guidelines for Screening Mutagenicity Testing of Chemicals (Nakajima, 2002). Properly maintained Chinese hamster lung (CHL) cells were treated with docosanoic acid dissolved in 1% carboxymethylcellulose sodium at concentrations of 875, 1750 and 3500 µg/mL for 6 hours with and without metabolic activation by S9-mix prepared from phenobarbital- and 5,6-benzoflavone-induced rat livers. The highest test concentration of 3500 µg/mL reflect 0.01 M of the test substance as required in the in OECD Guideline 473. In addition, the cells were incubated with 350, 700, 1400, 2800 µg/mL without metabolic activation for 24 hours and with 288, 575, 1150 and 2300 µg/mL without metabolic activation for 48 hours, respectively. The highest concentration of the test item used was set to the maximum one showing no apparent cytotoxic effects during continuous treatment. No increase in chromosomal aberrations nor polyploidy were observed up to the maximum concentration under short-term and continuous treatment with and without metabolic activation. The positive controls included during short-term and continuous exposure showed the expected results and thus verified the sensitivity of the assay.

Gene mutation in mammalian cells

No data on gene mutation in mammalian cells is available for docosanoic acid. Therefore the gene mutation potential in mammalian cells is predicted from adequate and reliable data for source substances by read-across to the target substance within the group applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. No mutagenic potential for fatty acids is expected since fatty acids are found in all living organisms where they are fulfilling fundamental physiological functions. This could be demonstrated by gene mutation assays in mammalian cells with C10 fatty acid (decanoic acid) and with C18 unsaturated fatty acids (linoleic and linolenic acid).

An in vitro mammalian cell gene mutation assay was performed with decanoic acid (CAS# 334-48-5) under GLP according to OECD Guideline 476 (Trenz, 2010). In two independent experiments, mouse lymphoma L5178Y cells were treated with decanoic acid at concentrations up to 1.18 mM without metabolic and up to 1.54 mM with metabolic activation by phenobarbital and beta-naphthoflavone-induced rat liver S9-mix, respectively. The exposure duration was 4 hours and 24 hours in experiments without S9 mix and 4 hours in the experiments with S9 mix. The treatment of cells in all experiments was followed by an expression period of 2 days and a selection period of 11-14 days in the presence of trifluorothymidine. Although cytotoxicity was observed at the highest concentrations tested, all mutant values were found to be within the range of the historical control data of the test facility, so that decanoic acid was regarded not to be mutagenic. In addition, colony sizing was performed for the highest concentrations used to detect potential clastogenic effects and/or chromosomal aberrations. As result, decanoic acid was found not to be clastogenic at all dose groups tested. The positive controls caused a pronounced increase in the mutation frequency demonstrating the sensitivity of the test system.

 

Another mouse lymphoma TK^+/-assay was performed with linoleic acid (CAS# 60-33-3) and linolenic acid (CAS# 463-40-1) similar to OECD Guideline 476 (Seifried et al., 2006). The applied test concentrations were very low and covered only a small dose range due to the insolubility of the test substances. Mouse lymphoma L5178Y cells were exposed for 4 hours to linoleic acid in a concentration range of 0.005 - 0.024 µL/mLwithout metabolic activation and to linoleic concentrations of 0.01 - 0.006 µL/mL with metabolic activation. Linolenic acid was tested in a concentration range of 0.021 - 0.025 µL/mL without S9 mix and in a range of 0.01 - 0.041 µL/mL with S9 mix. 2 days after treatment, treated cells were plated in soft agar medium containing TFT for 10 – 12 days. Cytotoxicity was observed for linoleic acid at the highest tested concentration with S9 mix (0.006 µL/mL). The relative total growth for linolenic aicd was decreased below 50% starting at 0.024 µL/mL without S9 mix and at 0.041 µL/mL with S9 mix. No increase in mutation frequency was observed for linoleic acid and linolenic acid compared to the control. Therefore, both C18 unsaturated fatty acids were considered not to be mutagenic in mammalian cells.

In summary, no gene mutation in mammalian cells was detected for different members of the category and consequently fatty acids are considered to be not genotoxic in vitro. Since all the members of the category show similar structural and toxicological properties the results of individual members are also valid for other fatty acids within the category.

Taking all the results together, the negative results of the available study data with docosanoic acid and different members of the category do not provide any evidence, that docosanoic acid is mutagenic or cytogenic as expected based on its physiological function within the body. 

Justification for classification or non-classification

All available data on genetic toxicity of the members of the fatty acids category do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.