(10R)-10-hydroxyoctadecanoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Oct - 10 Nov 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Principles of method if other than guideline:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Method

Target gene:

his operon; trp operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

First experiment (plate incorporation test): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate with and without metabolic activation
Second experiment (pre-incubation test): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Solvent: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (10 µg/plate) for TA100 and TA1535; 4-nitro-o-phenylene-diamine (10 µg/plate and 50 µg/plate) for TA1537 and TA98; methyl methane sulfonate (2 µl/plate) for WP2 uvrA; +S9: 2-aminoanthracene (2.5 µg/plate and 10 µg/plate) for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation; Experiment I); pre-incubation (Experiment II)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each of two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the number of spontaneous revertants and the bacterial background lawn

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduction on the number of revertants at 5000 µg/plate (Experiment I with and without S9 mix; Experiment II with S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduction on the number of revertants at 5000 µg/plate (Experiment I with and without S9 mix; Experiment II with S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

reduction on the number of revertants at 2500 - 5000 µg/plate (Experiment I with and without S9 mix; Experiment II with S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Test results 5

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 μg/plate in experiment I and from 1000 to 5000 μg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 1000 to 5000 μg/plate in both experiments. The undissolved particles had no influence on the data recording and the tested number of concentration levels that showed no precipitation was sufficient according to the guideline.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation)

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		Frameshift type		Base-pair substitutions and others
		TA 100	TA1535	TA98	TA1537	WP2 uvrA
–	DMSO	112 ± 9	9 ± 4	19 ± 2	7 ± 1	37 ± 3
–	Untreated	131 ± 8	9 ± 2	22 ± 6	8 ± 1	43 ± 1
–	3	111 ± 8	9 ± 3	20 ± 1	6 ± 3	32 ± 5
–	10	103 ± 8	8 ± 2	25 ± 8	8 ± 4	37± 7
–	33	113 ± 24	12 ± 3	23 ± 8	7 ± 0	46 ± 9
–	100	85 ± 8	9 ± 1	20 ± 1	8 ± 2	46 ± 6
–	333	76 ± 11	10 ± 2	27 ± 10	10 ± 4	42 ± 13
–	1000	91 ± 12 P	11 ± 1 P	17 ± 2 P	8 ± 2 P	57 ± 7 P
–	2500	49 ± 3 P	9 ± 2 P	19 ± 4 P	6 ± 1 P	39 ± 8 P
–	5000	46 ± 7 P	7 ± 2 P	6 ± 2 P	3 ± 1 P	30 ± 4 P
Positive controls, –S9	Name	NaN3	NaN3	4-NOPD	4-NOPD	MMS
	Concentrations (μg/plate)	10	10	10	50	2 [µL]
	Mean No. of colonies/plate (average of 3 ± SD)	2261 ± 165	1077 ± 131	382 ± 22	77 ± 8	988 ± 24
+	DMSO	87 ± 4	12 ± 4	24 ± 6	10 ± 2	47 ± 5
+	Untreated	127 ± 12	10 ± 2	34 ± 7	11 ± 3	49 ± 5
+	3	83 ± 4	13 ± 1	28 ± 7	9 ± 5	50 ± 5
+	10	86 ± 8	15 ± 2	24 ± 2	10 ± 2	40± 10
+	33	83 ± 4	12 ± 2	27 ± 6	8 ± 1	49 ± 8
+	100	65 ± 13	12 ± 4	29 ± 2	11 ± 4	37 ± 9
+	333	65 ± 13	12 ± 3	32 ± 10	9 ± 5	53 ± 11
+	1000	67 ± 12 P	15 ± 2 P	25 ± 3 P	10 ± 2 P	56 ± 8 P
+	2500	53 ± 3 P	9 ± 2 P	14 ± 3 P	6 ± 1 P	30 ± 3 P
+	5000	46 ± 2 P	9 ± 2 P	9 ± 1 P	2 ± 1 P	25 ± 4 P
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	2.5	2.5	10
	Mean No. of colonies/plate (average of 3 ± SD)	3458 ± 146	488 ± 30	301 ± 59	159 ± 11	370 ± 26

NaN₃ = Sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methane sulfonate

2AA = 2-Aminoanthracene

P = Precipitate

Table 2. Test results of experiment 2 (pre-incubation test)

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		Frameshift type		Base-pair substitutions and others
		TA 100	TA1535	TA98	TA1537	WP2 uvrA
–	DMSO	118 ± 3	10 ± 2	22 ± 5	11 ± 2	30 ± 10
–	Untreated	184 ± 21	9 ± 5	29 ± 5	8 ± 4	43 ± 6
–	3	111 ± 3	9 ± 3	23 ± 3	11 ± 2	35 ± 3
–	10	112 ± 22	10 ± 5	25 ± 5	10 ± 2	33 ± 9
–	33	112 ± 18	8 ± 3	23 ± 7	8 ± 3	44 ± 5
–	100	95 ± 16	10 ± 4	31 ± 2	9 ± 1	36 ± 7
–	333	60 ± 4	11 ± 3	21 ± 1	8 ± 2	45 ± 2
–	1000	60 ± 15 P	11 ± 3 P	24 ± 6 P	12 ± 0 P	35 ± 11 P
–	2500	45 ± 3 P	8 ± 1 P	19 ± 3 P	9 ± 2 P	34 ± 3 P
–	5000	44 ± 6 P	5 ± 2 P	22 ± 5 P	5 ± 1 P	33 ± 2 P
Positive controls, –S9	Name	NaN3	NaN3	4-NOPD	4-NOPD	MMS
	Concentrations (μg/plate)	10	10	10	50	2 [µL]
	Mean No. of colonies/plate (average of 3 ± SD)	1787 ± 97	938 ± 55	261 ± 39	90 ± 9	665 ± 51
+	DMSO	105 ± 11	10 ± 3	34 ± 1	13 ± 3	53 ± 12
+	Untreated	145 ± 22	10 ± 2	39 ± 9	15 ± 4	57 ± 8
+	3	116 ± 8	9 ± 3	41 ± 7	15 ± 2	44 ± 10
+	10	133 ± 16	10 ± 5	40 ± 2	17 ± 3	46 ± 8
+	33	112 ± 10	10 ± 5	40 ± 6	12 ± 3	47 ± 8
+	100	89 ± 8	11 ± 2	27 ± 9	12 ± 3	48 ± 15
+	333	85 ± 3	10 ± 3	23 ± 3	11 ± 3	52 ± 12
+	1000	93 ± 2 P	13 ± 3 P	27 ± 3 P	13 ± 2 P	55 ± 5 P
+	2500	92 ± 3 P	11 ± 2 P	20 ± 7 P	10 ± 1 P	40 ± 8 P
+	5000	85 ± 3 P	8 ± 1 P	13 ± 4 P	4 ± 1 P	27 ± 5 P
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	2.5	2.5	10
	Mean No. of colonies/plate (average of 3 ± SD)	2907 ± 251	350 ± 42	3610 ± 212	175 ± 21	270 ± 26

NaN₃ = Sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

MMS = methyl methane sulfonate

2AA = 2-Aminoanthracene

P = Precipitate

Table 3: Historical control data

Strain		- S9				+ S9
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA1535	Solvent control	16	2.84	7	27	18	4.50	7	36
	Untreated control	15	3.35	7	27	19	4.99	9	34
	Positive control	2248	468.15	803	3722	418	119.81	182	683
TA1537	Solvent control	10	2.19	6	23	18	4.37	8	32
	Untreated control	10	2.52	5	22	19	4.64	8	30
	Positive control	74	10.30	50	159	300	126.74	77	651
TA98	Solvent control	26	4.27	16	46	38	6.29	21	59
	Untreated control	28	5.15	16	54	41	6.40	21	55
	Positive control	326	56.74	211	560	2306	944.29	330	4691
TA100	Solvent control	107	20.07	74	184	132	22.97	83	205
	Untreated control	113	21.29	82	204	142	21.11	82	210
	Positive control	1987	376.71	478	2594	2864	946.03	530	4983
WP2uvrA	Solvent control	52	7.73	34	70	58	7.87	41	77
	Untreated control	52	7.91	32	81	57	7.71	39	74
	Positive control	716	259.60	226	1296	354	112.61	180	642

Mean = mean value if revertants/plate

SD = Standard Deviation

Min = minimal value

Max = maximal value