[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For the read-across justification see section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The test substance provided by NOF Corporation was used. The test substance was stored at room temperature until use. After the test was completed, the residual test substance was analyzed by the test substance provider, and as a result, there was no problem with stability.

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix was prepared from the liver of male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone as inducers.

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 µg/plate);Sodium azide(0.5 µg/plate);Aminoacridine hydrochloride(80 µg/plate);+S9: 2-Aminoanthracene(1.0 µg/plate;TA100),(2 µg/plate;TA1535;TA1537),(10 µg/plate;WP2 uvrA),(0.5 µg/plate;TA98)

Remarks:

These positive control substances were dissolved using DMSO, dispensed in small portions, and then cryopreserved (-20 ° C).

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37 °C
- Exposure duration: 48 hours; after that, the growth state of the test strain on the plate was observed using a stereomicroscope (× 60) in order to confirm the growth inhibitory effect of the test substance on the test strain

NUMBER OF REPLICATIONS: 2 times (3 plates/concentration/test)

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:

other: S. typhimurium TA 1535, TA 1537, TA 98 and 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

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

Conclusions:

Interpretation of results:
negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For the read-across justification see section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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 (LIFE TECHNOLOGIES)
- Properly maintained: yes

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% aqueous solution of sodium carboxymethyl cellulose (Wako Pure Chemical Industries, Ltd.)

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:

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

STAIN (for cytogenetic assays): Giemsa (1.2% for 12 min)

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. The frequency of occurrence of cells with chromosomal abnormalities was negative (-) for less than 5%, false positive (±) for 5% or more and less than 10%, and positive (+) for 10% or more. Ultimately, it was judged positive when reproducibility or dose dependence was observed.

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

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

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: negative.
The chromosomal aberration induction of docosanoic acid in cultured Chinese hamster cells was judged to be negative under the conditions of this test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

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)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

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:

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:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Remarks:

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

True negative controls validity:

not applicable

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)

Remarks on result:

other: all strains/cell types tested

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:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item decanoic acid is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available