Registration Dossier

Administrative data

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.

Link to relevant study records
Reference
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:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
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.

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.

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.