12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

The test chemical is not mutagenic to the Salmonella typhimurium strains in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Chromosome aberration assay:

The test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene Mutation Test-

Based on the data available, the test chemical is not mutagenic in mouse LS178Y mouse lymphoma cell colonies in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant 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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA97 and TA98

Remarks:

2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, and TA1537

Remarks:

3

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Remarks:

4

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Remarks:

4

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

2/3. 10% and 30% HLI and RLI S-9 (9,000 g supernatant) fractions were prepared from Aroclor 1254-induced, male Sprague- Dawley rat and male Syrian hamster livers

4. Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

2. 0, 0.1, 0.3, 1.0, 3.0, 6.7, 10.0, 33.0, 100.0, 200.0 µg/plate
3. 0, 3.3, 10, 33.0, 100, 333, 1000 or 3333 µg/plate
4. -S9 mix; 156, 313, 625, 1250, 2500 and 5000 ug/plate
+S9 mix; 156, 313, 625, 1250, 2500 and 5000 ug/plate

Vehicle / solvent:

2. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical is soluble in DMSO

3. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble and stable in DMSO

4. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance is soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (TA98; -S9) and 2-aminoanthracene (all strains; +S9)

Remarks:

2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene (2-AA) (All strains, +S9)

Remarks:

3

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide(TA100, TA98 and WP2 uvrA), Sodium azide(TA1535), 9-Aminoacridine hydrochloride (TA1537) +S9 mix; 2-Aminoanthracene(all strains)

Remarks:

4

Details on test system and experimental conditions:

2. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

3. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER: No data available

4. METHOD OF APPLICATION: Pre incubation method
NUMBER OF REPLICATIONS: Duplicate
OTHER EXAMINATIONS: 3 plates per test were observed.

Rationale for test conditions:

No data

Evaluation criteria:

2. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “ +W,” if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a nonmutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two fold over background for a chemical to be judged mutagenic. A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it produced a reproducible dose-related reponse over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or nonmutagenicity.

3. The chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials. It chemical was considered to be questionable (?) if a reproducible increase of his+ revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.

4. Evaluation was done considering a dose dependent increase in the number of revertants/plate

Statistics:

2. Mean ± SD
3. No data available
4. SD ± Mean

Species / strain:

S. typhimurium, other: TA100, TA1535, TA97 and TA98

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, and TA1537

Remarks:

3

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA100, TA1535, TA98, TA1537

Remarks:

4

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Remarks:

4

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

3. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: The chemical was tested initially in a toxicity assay to determine the appropriate dose range. The toxicity assay was performed by using TA100 or the system developed by Waleh et al. Toxic concentrations were those at which a decrease in the number of his+ colonies was seen or at which there was a clearing in the density of the background lawn

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

4. ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed up to a concentration of 5000 μg/plate with or without metabolic activation.

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical is not mutagenic to the Salmonella typhimurium strains in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of 12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide (CAS no 106 -15 -0). The studies are as mentioned below:

The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium. The test compound was dissolved in DMSO and was tested at concentration of 0, 0.1, 0.3, 1.0, 3.0, 6.7, 10.0, 33.0, 100.0, 200.0µg/plate using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study. The test chemical is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study for 80 -90% closely related test chemical chemical, Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs. The test compound was dissolved in DMSO and was used at a dosage level of 0, 3.3, 10, 33.0, 100, 333, 1000 or 3333 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study. The test chemical did not induce a reproducible, dose-related increase in his⁺revertants over the corresponding solvent in the S. typhimurium tester strains TA1535, TA1537, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is negative for mutation in vitro.

Genetic toxicity in vitro study was also assessed for test chemical. AMES test was performed according to OECD 471, 472 and Guidelines for Screening Mutagenicity Testing of Chemicals (Japan). The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 156, 313, 625, 1250, 2500 and 5000 µg/plate and DMSo was used as the solvent of choice. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non-mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Based on the data available, the test chemical is not mutagenic to the Salmonella typhimurium strains in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

6

Details on mammalian cell type (if applicable):

- Type and identity of media: McCoy’s 5A medium
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

7

Details on mammalian cell type (if applicable):

- Type and identity of media: Stocks of CHO
cells were maintained at 37°C in McCoy's 5A (modified) medium buffered with 20 mM
HEPES and supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 pg/ml streptomycin (Gibco. Grand Island,
NY).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes, Cells were tested regularly for mycoplasma contamination using 4' ,6-diamidin0-2-phenyl-indole (DAPI) fluorescence and were found to be free of mycoplasma
- Periodically checked for karyotype stability: Yes, To ensure karyotypic stability, cells were not used beyond the fifteenth passage after cloning.
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Remarks:

8

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

6./7. In the presence and absence of Aroclor 1254-induced male Sprague-Dawley rat liver S9 and cofactor mix
8. Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

6. 0, 16, 30 or 50 µg/mL
7. No data
8. -S9 mix(24hr continuous exposure): 0, 350, 700, 1400, 2800 μg/mL
-S9 mix(48hr continuous exposure): 0, 288, 575, 1150, 2300 μg/mL
-S9 mix(short-term exposure): 0, 875, 1750, 3500 μg/mL
+S9 mix(short-term exposure): 0, 875, 1750, 3500 μg/mL

Vehicle / solvent:

6. No data
7. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

8. - Vehicle(s)/solvent(s) used: 1% Carboxymethylcellulose sodium
- Justification for choice of solvent/vehicle: the test chemical was soluble in Carboxymethylcellulose sodium

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

No specified details

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

6

Untreated negative controls:

yes

Remarks:

Medium control

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

7

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

1% Carboxymethylcellulose sodium

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

8

Details on test system and experimental conditions:

6. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: Without S9: 12 hrs
With S9: 2 hrs
- Expression time (cells in growth medium): Without S9: 12 hrs, With S9: 13 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): Without S9: 12 hrs, With S9: 13 hrs

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: Two hundred first-division metaphase cells were scored at each dose level

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS: No data
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other:

OTHER: No data

7. METHOD OF APPLICATION: in medium

Cells seeded: 1.75 X 106 cells/75 cm2 flask

DURATION
- Preincubation period: No data
- Exposure duration: Without S9: 8 hrs
With S9: 2 hrs
- Expression time (cells in growth medium): Without S9: 10-10.5 hrs
With S9: 12 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 100-200 cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Other: No data

OTHER: No data

8. Chinese hamster lung(CHL)cells were used .

Rationale for test conditions:

No data

Evaluation criteria:

6. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations).

7. Selection of cells for scoring was based on well-spread chromosomes with good morphology and a chromosome number of 21 ± 2. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis.

A positive response at a single dose was designated “ + W”, weak evidence for clastogenicity. If there was a strong trend as the result of a large increase in ABs at a single dose only, we designated the result ‘‘ + W*”. A test was designated “ + ” if at least two doses gave significantly increased responses.

8. The cells were observed for chromosomal abbreviation, gaps.

Statistics:

6. Significance of percent cells with aberrations tested by the linear regression trend test versus log of the dose. To arrive at a statistical call for a trial, analyses were conducted on both the dose response curve and individual dose points. For a single trial, a statistically significant (P≤0.05) difference for one dose point and a significant trend (P≤0.015) were considered weak evidence for a positive response; significant differences for two or more doses indicated the trial was positive. A positive trend test in the absence of a statistically significant increase at any one dose resulted in an equivocal call

7. A binomial sampling assumption as described by Margolin et al. was used to examine absolute increases in ABs over solvent control levels at each dose. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. Only the “total” percent cells with aberrations were analyzed, and a positive response was defined as one for which the adjusted P value was <0.05.

8. No data

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

5

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO LB / 6

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

above 49.9 (-S9) and 30.3 (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: CHL/IU cells

Remarks:

8

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

5. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: the high dose was limited by toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

6. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: Test concentrations for the AB assays were empirically chosen based on toxicity and cell cycle delay as noted in the SCE experiments. At least five concentrations of the test chemical were selected; the concentrations were spaced using two merged half-log scales (e.g., 1,000, 500, 300, 150, 100, etc.), and the highest concentrations analyzed were those yielding a sufficient number of suitable metaphase cells. The concentrations analyzed generally covered a one-log range.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

7. Cytotoxicity conc.: With metabolic activation: None
Without metabolic activation:
>= 2,703 ug/mL (24 hr), 2,242 ug/mL (48 hr)

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity studies for the various test chemicals was reviewed to determine the mutagenic nature of the target chemical in vitro. The studies are as mentioned below:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the 60 -70% clasely related test chemical. The test chemical was dissolved in suitable solvent and used at dose levels of 0, 16, 30 or 50µg/mL using the Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. In the Abs test without S9, cells were incubated in McCoy’s 5A medium with coconut oil acid diethanolamine condensate for 10 hours; Colcemid was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with coconut oil acid diethanolamine condensate and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 11 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. The harvest time for the Abs test was based on the cell cycle information obtained in the SCE test. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). Based on the observations made, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of another 60 -70% closely related test chemical. Approximately 24 hr before chemical treatment, cultures were initiated at a density of 1.75 X 10⁶cells/75 cm²flask. In the AB trials without S9, the cultures were treated with the test chemical in medium for 8 hr, washed to remove the test chemical, and treated with colcemid M) for 2-2.5 hr before cell harvest. In the experiments with activation, cultures were exposed to the test chemical in serum free medium with S9 and cofactors for 2 hr, washed to remove the test chemical and S9, and incubated at 37°C with fresh medium for 8 hr. Colcemid was then added, andthe cells were harvested 2 hr later. Thus the total durations of the nonactivated and activated AB experiments were 10 hr and 12 hr, respectively, to give 10 hr growth in medium with serum for each experiment. For ABs, slides were stained in 5% Giemsa for 5 min. In early studies, one hundred cells were scored for each ofthree concentrations: the highest test concentration in whichsufficient metaphase cells could be scored and the next two lower concentrations, covering a one-log range. For later studies, 200 cells per dose were scored; however, fewer cells were scored if a test chemical produced a strong positive response or the chemical was toxic. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis. The test chemical did not induce chromosome aberrations in the CHO-LB cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Genetic toxicity in vitro study was also assessed for test chemical. For this purpose in vitro mammalian chromosome aberration test was performed according to OECD 473 and Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Chinese hamster lung (CHL) cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention below

-S9 mix(24hr continuous exposure): 0, 350, 700, 1400, 2800 µg/mL

-S9 mix(48hr continuous exposure): 0, 288, 575, 1150, 2300 µg/mL

-S9 mix(short-term exposure): 0, 875, 1750, 3500 µg/mL

+S9 mix(short-term exposure): 0, 875, 1750, 3500 µg/mL

 No chromosomal abbreviation or gaps were observed in the treated cells, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non-mutagenic in Chinese hamster lung (CHL) cells by in vitro mammalian chromosome aberration test. Hence the substance cannot be classified as gene mutant in vitro.

Based on the data available for the various test chemicals, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Remarks:

Read Across data

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the data from various test chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

no

Type of assay:

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

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

10

Details on mammalian cell type (if applicable):

L5178Y mouse lymphoma cells were maintained at 37° C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately10 hours.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

11

Details on mammalian cell type (if applicable):

mammalian cell line

Metabolic activation:

with and without

Metabolic activation system:

10. Metabolic activation system - S9 fraction in these experiments.
Type and composition of metabolic activation system:
- source of S9 : No data available
- method of preparation of S9 mix : Freshly prepared S9 from the livers of Aroclor 1254-induced male Fischer 344 rats.
- concentration or volume of S9 mix and S9 in the final culture medium : No data available

11. Metabolic activation system
Type and composition of metabolic activation system: Not specified
- source of S9 : No data available
- method of preparation of S9 mix : No data available
- concentration or volume of S9 mix and S9 in the final culture medium : No data available
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) :No data available

Test concentrations with justification for top dose:

10. The highest concentration was limited to 200 microgram /plate by toxicity.

11. 40-250 μg/mL (in DMSO) in the absence of metabolic activation; ranged from 10-100 μg/mL (in DMSO) in the presence of metabolic activation

Vehicle / solvent:

10. - Vehicle(s)/solvent(s) used: No data available
- Justification for choice of solvent/vehicle: No data available
- Justification for percentage of solvent in the final culture medium: No data available

11. - Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Test item was solubilized in DMSO
- Justification for percentage of solvent in the final culture medium: No data available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Remarks:

10

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: Ethylmethanesulfonate

Remarks:

11

Details on test system and experimental conditions:

10. L5178Y mouse lymphoma cells were maintained at 37° C as suspension cultures in supplemented Fischer's medium; nonnal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring cells resistant to trifluorothymidine (TFT), subcultures were exposed to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added.

All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 X 106 cells in 10 mL medium. This volume included the S9
fraction in those experiments perfonned with metabolic activation. Incubation with coconut oil acid diethanolamine condensate continued for 4 hours, at which time the medium plus coconut oil acid
diethanolamine condensate was removed, and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells, and cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37° C in 5% CO2 for 10 to 12 days.

11. NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : No data available
- Number of independent experiments : No data available
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data available
- Test substance added in medium; in agar (plate incorporation); preincubation.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No data available
- Exposure duration/duration of treatment: No data available
- Harvest time after the end of treatment (sampling/recovery times): No data available
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): No data available
- Selection time (if incubation with a selective agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available
- Method used: agar plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: No
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: No data
available
- Criteria for small (slow growing) and large (fast growing) colonies: No data available
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: No data available
- Any supplementary information relevant to cytotoxicity: No data available
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- OTHER: No data available

Evaluation criteria:

10. A single significant response led to a "questionable" conclusion, and the absence of both a trend and peak response resulted in a "negative" call.

11. Not specified

Statistics:

10. All data were evaluated statistically for trend and peak responses.

11. Not specified

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

10

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

11

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

10. TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data available
- Data on osmolality: No data available
- Possibility of evaporation from medium: No data available
- Water solubility: No
- Precipitation and time of the determination: No data available
- Definition of acceptable cells for analysis: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES (if applicable):

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Yes

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: No data available
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: No data available

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures : 6 X 106 cells
o Number of cells plated in selective and non-selective medium : No data available
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency : No data available
o When using the thymidine kinase gene on L5178Y cells: No data available.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: No data available
- Negative (solvent/vehicle) historical control data: No data available

11. TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data available
- Data on osmolality: No data available
- Possibility of evaporation from medium: No data available
- Water solubility: No data available
- Precipitation and time of the determination: No data available
- Definition of acceptable cells for analysis: No data available
- Other confounding effects: No data available
RANGE-FINDING/SCREENING STUDIES (if applicable): No data available
STUDY RESULTS
- Concurrent vehicle negative and positive control data : No data available
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency
- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures : No data available
o Number of cells plated in selective and non-selective medium : No data available
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency : No data available
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: No data available
- Negative (solvent/vehicle) historical control data: No data available

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical, 12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide (CAS no 106 -15 -0) is not mutagenic in mouse LS178Y mouse lymphoma cell colonies in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of 12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide (CAS no 106 -15 -0). The studies are as mentioned below:

The study was conducted on L5178Y mouse lymphoma cells to determine Genetic toxicity of 12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide. L5178Y mouse lymphoma cells were maintained at 37° C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 X 106 cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with coconut oil acid diethanolamine condensate continued for 4 hours, at which time the medium plus coconut oil acid diethanolamine condensate was removed, and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells, and cells were plated in nonselective medium and soft agar to determine cloning efficiency. To reduce the number of spontaneously occurring cells resistant to trifluorothymidine (TFT), subcultures were exposed to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added. No increase in mutant LS178Y mouse lymphoma cell colonies was observed after exposure to coconut oil acid diethanolamine condensate, with or without S9. A single positive response noted at 8 nL/mL in the second trial conducted without S9 was not reproducible and the test results overall were considered to be negative.

In another study Hydroxystearic acid (CAS: 106 -14 -9) was tested for in vitro mammalian cell gene mutation assay test using mouse lymphoma L5178Y cells to determinine the mutagenicity. The test was performed with and without metabolic activation system using rat liver homogenate. The rat liver homogenate was obtained from Aroclor-1254- induced Wistar rats.Dimethyl sulfoxide (DMSO) was selected as a vehicle. The doses ranged from 40-250 µg/mL (in DMSO) in the absence of metabolic activation; ranged from 10-100 µg/mL (in DMSO) in the presence of metabolic activation. The cultures treated with doses 250-40µg/mL (non-activated) showed total growth from 1-71% and the cultures treated with doses 100-10 µg/mL (activated) showed 18-119% total growth. No dose dependent response was observed in activated or non-activated cultures. The solvent and positive controls fulfilled the requirements for a vaild test. Hence, the test substanceHydroxystearic acid (CAS: 106 -14 -9)was found to be non-mutagenic in the presence or absence of metabolic activation system at anyof the concentrationsinmouse lymphoma L5178Y cells.

Based on the data available the test chemical, 12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide (CAS no 106 -15 -0) is not mutagenic in mouse LS178Y mouse lymphoma cell colonies in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

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 vitro:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of 12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide (CAS no 106 -15 -0). The studies are as mentioned below:

Ames test:

The test chemical was studied for its ability to induce mutations in strains of Salmonella typhimurium. The test compound was dissolved in DMSO and was tested at concentration of 0, 0.1, 0.3, 1.0, 3.0, 6.7, 10.0, 33.0, 100.0, 200.0µg/plate using Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of 10 % and 30 % rat and hamster liver S9 metabolic activation system. Preincubation assay was performed with a preicubation for 20 mins. The plates were observed for histidine independence after 2 days incubation period. Concurrent solvent and positive controls were included in the study. The test chemical is not mutagenic to the Salmonella typhimurium TA100, TA1535, TA97 and TA98 in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study for 80 -90% closely related test chemical chemical, Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of the test chemical usingS. typhimuriumtester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs. The test compound was dissolved in DMSO and was used at a dosage level of 0, 3.3, 10, 33.0, 100, 333, 1000 or 3333 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study. The test chemical did not induce a reproducible, dose-related increase in his⁺revertants over the corresponding solvent in theS. typhimuriumtester strains TA1535, TA1537, TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence is negative for mutation in vitro.

Genetic toxicity in vitro study was also assessed for test chemical. AMES test was performed according to OECD 471, 472 and Guidelines for Screening Mutagenicity Testing of Chemicals (Japan). The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 156, 313, 625, 1250, 2500 and 5000 µg/plate and DMSO was used as the solvent of choice. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non-mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

Chromosome aberration assay:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the 60 -70% clasely related test chemical. The test chemical was dissolved in suitable solvent and used at dose levels of 0, 16, 30 or 50µg/mL using the Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system. In the Abs test without S9, cells were incubated in McCoy’s 5A medium with coconut oil acid diethanolamine condensate for 10 hours; Colcemid was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with coconut oil acid diethanolamine condensate and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 11 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. The harvest time for the Abs test was based on the cell cycle information obtained in the SCE test. Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes). All slides were scored blind and those from a single test were read by the same person. Two hundred first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). Based on the observations made, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of another 60 -70% closely related test chemical. Approximately 24 hr before chemical treatment, cultures were initiated at a density of 1.75 X 10⁶cells/75 cm²flask. In the AB trials without S9, the cultures were treated with the test chemical in medium for 8 hr, washed to remove the test chemical, and treated with colcemid M) for 2-2.5 hr before cell harvest. In the experiments with activation, cultures were exposed to the test chemical in serum free medium with S9 and cofactors for 2 hr, washed to remove the test chemical and S9, and incubated at 37°C with fresh medium for 8 hr. Colcemid was then added, andthe cells were harvested 2 hr later. Thus the total durations of the nonactivated and activated AB experiments were 10 hr and 12 hr, respectively, to give 10 hr growth in medium with serum for each experiment. For ABs, slides were stained in 5% Giemsa for 5 min. In early studies, one hundred cells were scored for each ofthree concentrations: the highest test concentration in whichsufficient metaphase cells could be scored and the next two lower concentrations, covering a one-log range. For later studies, 200 cells per dose were scored; however, fewer cells were scored if a test chemical produced a strong positive response or the chemical was toxic. Cells were analyzed for the following categories of chromosomal aberrations: “simple,” defined as a chromatid gap, break, fragment, and deletion or chromosome gap,break, or double minutes; “complex,” defined as interstitial deletions, triradials, quadriradials, rings, and dicentric chromosomes; and “other,” defined as pulverized chromosomesor cells with greater than 10 aberrations. Chromatid and chromosome gaps were recorded but were not used in the analysis. The test chemical did not induce chromosome aberrations in the CHO-LB cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Genetic toxicity in vitro study was also assessed for test chemical. For this purpose in vitro mammalian chromosome aberration test was performed according to OECD 473 and Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Chinese hamster lung (CHL) cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention below

-S9 mix(24hr continuous exposure): 0, 350, 700, 1400, 2800 µg/mL

-S9 mix(48hr continuous exposure): 0, 288, 575, 1150, 2300 µg/mL

-S9 mix(short-term exposure): 0, 875, 1750, 3500 µg/mL

+S9 mix(short-term exposure): 0, 875, 1750, 3500 µg/mL

 No chromosomal abbreviation or gaps were observed in the treated cells, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non-mutagenic in Chinese hamster lung (CHL) cells by in vitro mammalian chromosome aberration test. Hence the substance cannot be classified as gene mutant in vitro.

Based on the data available for the various test chemicals, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available and applying the weight of evidence approach, the test chemical 12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide (CAS no 106 -15 -0) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned n CLP regulation.

Gene Mutation Assay

Data available for the various test chemicals was reviewed to determine the mutagenic nature of 12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide (CAS no 106 -15 -0). The studies are as mentioned below:

The study was conducted on L5178Y mouse lymphoma cells to determine Genetic toxicity of 12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide. L5178Y mouse lymphoma cells were maintained at 37° C as suspension cultures in supplemented Fischer's medium; normal cycling time was approximately 10 hours. All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 X 106 cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with coconut oil acid diethanolamine condensate continued for 4 hours, at which time the medium plus coconut oil acid diethanolamine condensate was removed, and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells, and cells were plated in nonselective medium and soft agar to determine cloning efficiency. To reduce the number of spontaneously occurring cells resistant to trifluorothymidine (TFT), subcultures were exposed to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added.

No increase in mutant LS178Y mouse lymphoma cell colonies was observed after exposure to coconut oil acid diethanolamine condensate, with or without S9. A single positive response noted at 8 nL/mL in the second trial conducted without S9 was not reproducible and the test results overall were considered to be negative.

In another study Hydroxystearic acid (CAS: 106 -14 -9) was tested for in vitro mammalian cell gene mutation assay test using mouse lymphoma L5178Y cells to determinine the mutagenicity. The test was performed with and without metabolic activation system using rat liver homogenate. The rat liver homogenate was obtained from Aroclor-1254- induced Wistar rats.Dimethyl sulfoxide (DMSO) was selected as a vehicle.

The doses ranged from 40-250 µg/mL (in DMSO) in the absence of metabolic activation; ranged from 10-100 µg/mL (in DMSO) in the presence of metabolic activation.

The cultures treated with doses 250-40µg/mL (non-activated) showed total growth from 1-71% and the cultures treated with doses 100-10 µg/mL (activated) showed 18-119% total growth.No dose dependent response was observed in activated or non-activated cultures. The solvent and positive controls fulfilled the requirements for a vaild test.

Hence, the test substanceHydroxystearic acid (CAS: 106 -14 -9)was found to be non-mutagenic in the presence or absence of metabolic activation system at anyof the concentrationsinmouse lymphoma L5178Y cells.

Based on the data available, the test chemical is not mutagenic in mouse LS178Y mouse lymphoma cell colonies in the presence and absence of rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available and applying the weight of evidence approach, the test chemical 12-hydroxy-N-(2-hydroxyethyl)octadecan-1-amide (CAS no 106 -15 -0) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned n CLP regulation.