Octadecyl stearate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

 

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence andabsence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

 

In vitro gene mutation study in mammalian cells

Test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation 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 of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE for the target CAS is summarized based on 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: TA98, TA100, TA1535, TA1537 and TA1538

Remarks:

2.

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2

Remarks:

2.

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Remarks:

3.

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

2.10% Aroclor 1254-induced liver S9 from male Sprague-Dawley rats.
3.Liver homogenates (S-9 fraction) from Aroclor 1254-induced male Sprague-Dawley rats (RLI) or male Syrian hamsters (HLI)

Test concentrations with justification for top dose:

2. 0, 0.033, 0.10, 0.33, 1.0, 3.3, 10 mg per plate
3. 0, 100, 333, 1000, 3333, 10000 µg / plate

Vehicle / solvent:

2.- Vehicle(s)/solvent(s) used: 0.067 M potassium or sodium phosphate buffer, pH 7.0
- Justification for choice of solvent/vehicle: The chemical was soluble in potassium or sodium phosphate buffer

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

Untreated negative controls:

not specified

Remarks:

2.

Negative solvent / vehicle controls:

yes

Remarks:

0.067 M potassium or sodium phosphate buffer, pH 7.0

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

furylfuramide

other: Anthramine (All strains; with S9)

Untreated negative controls:

not specified

Remarks:

3.

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (for TA98; without S9) and 2-amino-anthracene (2 AA; with S9)

Details on test system and experimental conditions:

2. METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- 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): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: All platings were performed in duplicate and all tests were
repeated.

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data

3. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 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): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: All tests were repeated atleast once

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

2. Test results were considered valid only if the positive control compounds induced increases in mutant counts to at least twice background.
3.A mutagenic response was defined as a reproducible, dose-related increase in the number of histidine-independent colonies over the spontaneous incidence; there was no requirement for a specific magnitude of increase.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

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:

E. coli WP2

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: TA100, TA1535, TA98, 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

Additional information on results:

2. No data

3. 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 final dose level selection was based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S-9.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce mutation in the Salmonella typhimurium and E. coli strains with and without rat and hamster liver S9 mix and hence the chemical is negative for gene mutation in vitro.

Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed as per the plate incorporation protocol and the test chemical dissolved in0.067 M potassium or sodium phosphate buffer, pH 7.0 was used at dose levels of 0, 0.033, 0.10, 0.33, 1.0, 3.3, 10 mg per plate. Concurrent solvent and positive controls were run with the test chemical. Test results were considered valid only if the positive control compounds induced increases in mutant counts to at least twice background. The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli strain WP2 with and without rat liver S9 mix and hence the chemical is negative for gene mutation in vitro.

 

In another study, bacterial reverse mutation assay was performed for the test chemical using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 with and without rat and hamster liver S9 mix. The study was performed using the preincubation protocol at five dose from 0, 100, 333, 1000, 3333, 10000µg / plate with acetone as the solvent control with incubation period of 48 hrs in the presence and absence of S9 mix.The final dose level selection was based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S-9. No mutagenic response was noted for the test compound in the preliminary dose range finding study and the main study performed. The test chemical did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA98, TA1537 with and without rat and hamster liver S9 mix and hence the chemical is negative for gene mutation in vitro.

 

Based on the observations made, the test chemical did not induce mutation in the Salmonella typhimurium and E. coli strains with and without rat and hamster liver S9 mix and hence the chemical is negative for gene mutation 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 various test chemicals.

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 for the target CAS is summarized based on 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 lung fibroblasts (V79)

Remarks:

5.

Details on mammalian cell type (if applicable):

- Type and identity of media: Inactivated (56 ° C., 30 minutes) calf serum which had been filtered under pressure byusing a membrane filter (0.45 μm) was added to Eagle-MEM liquid medium
at a final concentration To 10%, and then used for the test. The prepared culture solution was stored in a cool dark place (4 ° C.).
- Properly maintained: Yes, Cells were cultured at a CO2 concentration of 5% and 37 °C using a CO2 incubator
- 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 lung fibroblasts (V79)

Remarks:

6.

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

5.S9 mix was prepared from the liver of Sprague-Dawley male rats administered phenobarbital and 5,6-benzoflavone as inducing agents
6.The S9 liver microsomal fraction was obtained from the livers of male Wistar Hanlbm and was prepared by the testing facility CCR.

Test concentrations with justification for top dose:

5. 0, 1188, 2375 or 4750 µg/mL
6. 0, 0.6, 0.75, 1.5, 3.0, 5.0, 6.0, 10.0, 20.0, 30.0, 40.0 and 300.0 µg/ml

Vehicle / solvent:

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

6. - Vehicle(s)/solvent(s) used: No data
- Justification for choice of solvent/vehicle: No data

Untreated negative controls:

not specified

Remarks:

5.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Untreated negative controls:

not specified

Remarks:

6.

Negative solvent / vehicle controls:

no

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

5. METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: No data
- Exposure duration: 24 ad 48 hrs
- Expression time (cells in growth medium): 24 ad 48 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): 1.2% Giemsa

NUMBER OF REPLICATIONS: One hundred per plate, that is, 200 metaphase images per dose, was observed

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data

6. Details on test system and conditions
METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: No data
- Exposure duration: Experiment 1: 4 hrs (with and without metabolic activation)
Experiment 2: 18 and 28 hrs (without metabolic activation)
- Expression time (cells in growth medium): No data
- 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): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 100 metaphase plates were scored for chromosome aberrations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Test article concentrations between 1 and 300 ug/ml (-S9) or 1 and 285 ug/ml (+S9) were chosen for the assessment of the cytotoxic potential.

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

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

5. The cell line was observed for the gaps, chromatid breaks (ctb), chromosome breaks (csb). They were classified as structural anomalies of exchange (cte), chromosome exchange (cse) and others (oth). At the same time, the incidence of ploidy cells was recorded.

The frequency of occurrence of chromosomal structural abnormality was distinguished into cases where the cells possessing only the gap (+ gap) were included and when not included (-gap).
Frequency of occurrence of cells having structural abnormality in each test group or ploidy cells was judged according to the criteria of Ishikan et al. Negative (-) for cells with chromosomal abnormality negative (-), false positive (±) from 5% to less than 10%, positive (+) above 10%. Ultimately, it was judged as positive when reproducibility or dose dependence was observed.

6.In each experimental group two parallel cultures were set up. Per culture, 100 metaphase plates were scored for chromosome aberrations. The cell line was observed for chromosome aberration.

Statistics:

5.Chromosome analysis was carried out according to the classification method by Japan Environmental Mutagen Society and Mammal Test Subcommittee 2.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

5.

Metabolic activation:

with and without

Genotoxicity:

not specified

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 lung fibroblasts (V79)

Remarks:

6.

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

With and without S9 mix, in both experiments, no reduction of the mitotic index or the cell number was observed, except in the presence of S9 mix in experiment II at interval 28 h after treatment with 40 ug/ml reduced cell numbers were observed.

Vehicle controls validity:

valid

Untreated 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: Cells were seeded on a multi-plate for cell culture, and the test substance solution was treated 3 days after the culture. In the case of continuous treatment, the treatment is continuously carried out for 24 or 48 hours, in the case of the short time treatment, treatment is carried out for 6 hours in the absence of S9 mix (-S9 mix) or in the presence (+ S 9 mix), and then fresh culture medium, and the culture was continued for further 18 hours.
After fixing the cells with 10% neutral buffered formalin solution, the cells were stained with 0.1% Crystal Violet aqueous solution for 10 minutes. An appropriate amount of pigment eluate (30% ethanol, 1% acetic acid aqueous solution) was added and allowed to stand for about 5 minutes to elute the dye, and the absorbance at 580 nm was measured. For each dose group, the ratio to the absorbance in the solvent control group, ie the cell viability was calculated. As a result, clear cell growth suppression was not observed in any treatment method

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: Yes, pre-experiment was performed.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

5.

Table: Chromosomal aberration study (continuous exposure)

Compound	Dose (µg/mL)	Time (hr)	No. of cells analuysed	No. of cells with structural aberrations						Total (+gap) (%)	Total (-gap) (%)	Polyploid cells (%)	Final judgement
				Gap	Ctb	Csb	Cte	Cse	other				SA	POL
Test chemical	0	24	200	1	1	0	1	0	0	1.0	0.5	0.0	-	-
	1188	24	200	0	0	0	1	0	0	0.5	0.5	1.5	-	-
	2375	24	200	1	1	0	3	0	0	2.0	2.0	1.0	-	-
	4750	24	200	1	1	1	3	0	0	2.5	2.0	0.0	-	-
MMC	0.05	24	200	21	51	0	94	1	0	61.5	57.0	0.5	+	-
Test chemical	0	48	200	2	0	0	0	0	0	1.0	0.0	0.5	-	-
	1188	48	200	0	1	0	2	0	0	1.5	1.5	2.0	-	-
	2375	48	200	3	1	0	2	0	0	2.5	1.5	0.5	-	-
	4750	48	200	2	2	1	0	0	0	2.0	1.0	0.0	-	-
MMC	0.025	48	200	31	63	2	97	2	1	64.0	61.5	1.0	+	-

 

Table: Chromosomal aberration study (short term treatment exposure)

Compound	Dose (µg/mL)	Time (hr)	No. of cells analuysed	No. of cells with structural aberrations						Total (+gap) (%)	Total (-gap) (%)	Polyploid cells (%)	Final judgement
				Gap	Ctb	Csb	Cte	Cse	other				SA	POL
Test chemical	0	6	200	1	0	0	1	0	0	1.0	0.5	0.0	-	-
	1188	6	200	1	0	0	0	0	0	0.5	0.0	1.0	-	-
	2375	6	200	0	0	0	2	0	0	1.0	1.0	1.0	-	-
	4750	6	200	5	1	0	0	0	0	3.0	0.5	0.5	-	-
CP	12.5	6	200	2	1	0	0	0	0	1.5	0.5	0.5	+	-
Test chemical	0	6	200	1	0	0	1	0	0	1.0	0.5	0.5	-	-
	1188	6	200	0	0	0	1	0	0	0.5	0.5	1.0	-	-
	2375	6	200	0	2	0	4	0	0	2.0	2.0	1.5	-	-
	4750	6	200	0	0	0	3	0	0	1.5	1.5	0.5	-	-
CP	12.5	6	200	17	56	0	155	2	1	84.5	83.5	0.0	+	-

Conclusions:

The test chemical did not induce chromosome aberration in Chinese hamster lung derived fibroblast cell line with and without S9 metabolic activation system and hence the chemical is negative for gene mutation in vitro.

Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster lung derived fibroblast cell line (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 1188, 2375 or 4750 µg/mL. The study for performed for 24 and 48 hrs time period. The cell line was observed for the gaps, chromatid breaks (ctb), chromosome breaks (csb), chromosome segments (csb), chromosome segments. They were classified as structural anomalies of exchange (cte), chromosome exchange (cse) and others (oth). At the same time, the incidence of ploidy cells was recorded. The test chemical did not induce chromosome aberration in Chinese hamster lung derived fibroblast cell line (CHL) with and without S9 metabolic activation system and hence the chemical is negative for gene mutation in vitro.

 

In another study, in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using V79 cells in the presence and absence of S9 metabolic activation system. The test chemical was used at dose level of 0, 0.6, 0.75, 1.5, 3.0, 5.0, 6.0, 10.0, 20.0, 30.0, 40.0 and 300.0µg/mL. The study was performed for 4 hrs in experiment 1 for 18 hrs and 28 hrs time period in experiment 2. The cell line was observed for chromosome aberrations. The S9 liver microsomal fraction was obtained from the livers of male Wistar Hanlbm (BRL, CH-4414). The highest applied concentration in the pre-test was chosen with regard to the properties of the formulation of the test article. A homogenous suspension could be prepared at 300 ug/ml in the absence of S9 mix and 285 ug/ml in the presence of S9 mix. Test article concentrations between 1 and 300 ug/ml (-S9) or 1 and 285 ug/ml (+S9) were chosen for the assessment of the cytotoxic potential. In the absence of S9 mix reduced cell numbers to 55% of control were observed after treatment with 300 uglml whereas in the presence of S9 mix no toxic effects were observed. In the pre-test precipitation of the test article was observed 4 h after start of treatment at concentrations of 5 ug/ml and above in the absence of S9 mix and 10 ug/ml and above in the presence of S9 mix. In both independent experiments, neither a significant no a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test article.In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.Appropriate mutagens (Ethylmethane sulfonate and Cyclophosphamide) were used as positive controls. They induced statistically significant increase in cells with structural chromosome aberrations.  Thus,The test chemical did not induce chromosome aberration in V79 vells with and without S9 metabolic activation system and hence the chemical is negative for gene mutation in vitro.

 

Based on the observations made, the test chemical did not induce chromosome aberration in Chinese hamster lung derived fibroblast cell line with and without S9 metabolic activation system and hence the chemical is negative for gene mutation 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)

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 of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals i.e. WoE-8 and WoE-9.

GLP compliance:

not specified

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

8.Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

9.Thymidine kinase

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

8.

Details on mammalian cell type (if applicable):

- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

9.

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

8.S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

8. 0, 1, 2.5, 5 or 10 mM
9. Not specified but the chemical was tested at cytotoxic contrations according to the authors.

Vehicle / solvent:

8.- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/ vehicle: Test chemical was dissolved in ethanol.

9.Not specified.

Untreated negative controls:

yes

Remarks:

8.

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: N-ethyl-N-nitrosourea (ENU) (Without S9)

Untreated negative controls:

not specified

Remarks:

9.

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

8.METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Preincubation period: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable

9.Not specified

Rationale for test conditions:

No data

Evaluation criteria:

8.Dose dependent increase in the gene mutation activity
9.Criteria for transformation: Randomly oriented three-dimensional growth with extensive crossing over of the cells at the periphery of the colony was considered to be the endpoint of morphological transformation. The centres of transformed colonies usually exhibit dense piling-up of cells. Moreover, these cells usually have an increased ratio of nucleus to cytoplasm, are more basophilic, and are variable in size.

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

8.

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

8.

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

5 mM or less

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

9.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

no

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: no mutagenic potential

8.

Table 1A.Effect of test chemical exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO₂. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO₂. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

	With S9		Without S9
	with TG	without TG	with TG	without TG
Neg. control	2/4^a	195/4	0/4	194/4
Pos. control	0/4	210/4	13/4	124/4
1.0 mM	0/4	202/4	0/4	200/4
2.5 mM	0/4	181/4	0/4	156/4
5.0 mM	0/4	163/4	0/4	145/4
10.0 mM	20/4	92/4	0/4	153/4

 

^a)Two very diffuse colonies were found in one single well.

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to test chemical in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

	With S9	Without S9
Neg. control	N/A^a	N/A
Pos. control	N/A	3.43x10^-4
1.0 mM	N/A	N/A
2.5 mM	N/A	N/A
5.0 mM	N/A	N/A
10.0 mM	5.13 x10^-4	N/A

 

^a)Since two very diffuse colonies were found in one single well (see Table 1A), the diffuse colonies was not regarded as reliable and true colonies since the cells seemed to be apoptotic.

Conclusions:

Test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of test chemical when administered to Chinese Hamster Ovary (CHO) cells.In the genotoxicity test, zinc distearate was administered to CHO cells for 3 hrs at the dose levels of 0, 1.0, 2.5, 5.0 or 10.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four for the negative control in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the two spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. The presence of several colonies were observed in cells treated with 10.0 mM in the presence with S9 liver microsomal fraction. When the mutation frequency was determined, a frequency of 3.43 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, while exposure to 10.0 mM zinc stearate in the presence of S9 liver microsomal fraction showed a frequency of 5.13 x 10-4.The results indicate that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 5.0 mM, but when exposed to concentrations above 5.0 mM for 3 hrs, and in organisms with a fully functioning metabolic activation, the test chemical may induce gene mutations. When treated with test chemical and in the absence with S9 liver microsomal fraction, the results show no evidence of genotoxicity when CHO cells are exposed to concentrations of 1.0, 2.5, 5.0 or 10.0 mM, thus indicating that the test chemical does not give rise to gene mutations in organisms who have no or non-functional metabolic activation at the above mentioned concentrations.

 

Conclusion:

With S9 metabolic activation:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment with test chemical. The results showed evidence of gene toxicity when cells were exposed to test chemical at 10 mM, while treatment with 5 mM or less did not cause gene toxicity. Therefore, it is considered that test chemical in the concentrations of 5 mM or below does not cause genetic mutation(s), while concentrations at 10 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation.

 

Without S9 metabolic activation:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and without metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment with the test chemical. Independently of treatment concentration of test chemical, the results showed no evidence of potential gene toxicity. Therefore, it is considered that test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation.

 

In another study the test chemical was tested for mutagenicity in L5178Y mouse lymphoma cells. In the study, the test chemical was tested with and without metabolic activation up to cytotoxic doses.Test chemical was concluded to be non-mutagenic in mouse lymphoma cells.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation 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 from various sources was reviewed to determine the mutagenic nature of the given test chemical. The studies are as mentioned below:

 

Ames assay:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed as per the plate incorporation protocol and the test chemical dissolved in0.067 M potassium or sodium phosphate buffer, pH 7.0 was used at dose levels of 0, 0.033, 0.10, 0.33, 1.0, 3.3, 10 mg per plate. Concurrent solvent and positive controls were run with the test chemical. Test results were considered valid only if the positive control compounds induced increases in mutant counts to at least twice background. The test chemical did not induce mutation in the Salmonella typhimurium strain TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli strain WP2 with and without rat liver S9 mix and hence the chemical is negative for gene mutation in vitro.

 

In another study, bacterial reverse mutation assay was performed for the test chemical using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 with and without rat and hamster liver S9 mix. The study was performed using the preincubation protocol at five dose from 0, 100, 333, 1000, 3333, 10000µg / plate with acetone as the solvent control with incubation period of 48 hrs in the presence and absence of S9 mix.The final dose level selection was based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S-9. No mutagenic response was noted for the test compound in the preliminary dose range finding study and the main study performed. The test chemical did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA98, TA1537 with and without rat and hamster liver S9 mix and hence the chemical is negative for gene mutation in vitro.

 

Based on the observations made, the test chemical did not induce mutation in the Salmonella typhimurium and E. coli strains with and without rat and hamster liver S9 mix and hence the chemical is negative for gene mutation in vitro.

 

In vitro mammalian chromosome aberration study:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster lung derived fibroblast cell line (CHL) in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 1188, 2375 or 4750 µg/mL. The study for performed for 24 and 48 hrs time period. The cell line was observed for the gaps, chromatid breaks (ctb), chromosome breaks (csb), chromosome segments (csb), chromosome segments. They were classified as structural anomalies of exchange (cte), chromosome exchange (cse) and others (oth). At the same time, the incidence of ploidy cells was recorded. The test chemical did not induce chromosome aberration in Chinese hamster lung derived fibroblast cell line (CHL) with and without S9 metabolic activation system and hence the chemical is negative for gene mutation in vitro.

 

In another study, in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using V79 cells in the presence and absence of S9 metabolic activation system. The test chemical was used at dose level of 0, 0.6, 0.75, 1.5, 3.0, 5.0, 6.0, 10.0, 20.0, 30.0, 40.0 and 300.0µg/mL. The study was performed for 4 hrs in experiment 1 for 18 hrs and 28 hrs time period in experiment 2. The cell line was observed for chromosome aberrations. The S9 liver microsomal fraction was obtained from the livers of male Wistar Hanlbm (BRL, CH-4414). The highest applied concentration in the pre-test was chosen with regard to the properties of the formulation of the test article. A homogenous suspension could be prepared at 300 ug/ml in the absence of S9 mix and 285 ug/ml in the presence of S9 mix. Test article concentrations between 1 and 300 ug/ml (-S9) or 1 and 285 ug/ml (+S9) were chosen for the assessment of the cytotoxic potential. In the absence of S9 mix reduced cell numbers to 55% of control were observed after treatment with 300 uglml whereas in the presence of S9 mix no toxic effects were observed. In the pre-test precipitation of the test article was observed 4 h after start of treatment at concentrations of 5 ug/ml and above in the absence of S9 mix and 10 ug/ml and above in the presence of S9 mix. In both independent experiments, neither a significant no a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test article.In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls.Appropriate mutagens (Ethylmethane sulfonate and Cyclophosphamide) were used as positive controls. They induced statistically significant increase in cells with structural chromosome aberrations.  Thus,The test chemical did not induce chromosome aberration in V79 vells with and without S9 metabolic activation system and hence the chemical is negative for gene mutation in vitro.

 

Based on the observations made, the test chemical did not induce chromosome aberration in Chinese hamster lung derived fibroblast cell line with and without S9 metabolic activation system and hence the chemical is negative for gene mutation in vitro.

 

In vitro gene mutation study in mammalian cells:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of test chemical when administered to Chinese Hamster Ovary (CHO) cells.In the genotoxicity test, zinc distearate was administered to CHO cells for 3 hrs at the dose levels of 0, 1.0, 2.5, 5.0 or 10.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four for the negative control in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the two spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. The presence of several colonies were observed in cells treated with 10.0 mM in the presence with S9 liver microsomal fraction. When the mutation frequency was determined, a frequency of 3.43 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, while exposure to 10.0 mM zinc stearate in the presence of S9 liver microsomal fraction showed a frequency of 5.13 x 10-4.The results indicate that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 5.0 mM, but when exposed to concentrations above 5.0 mM for 3 hrs, and in organisms with a fully functioning metabolic activation, the test chemical may induce gene mutations. When treated with test chemical and in the absence with S9 liver microsomal fraction, the results show no evidence of genotoxicity when CHO cells are exposed to concentrations of 1.0, 2.5, 5.0 or 10.0 mM, thus indicating that the test chemical does not give rise to gene mutations in organisms who have no or non-functional metabolic activation at the above mentioned concentrations.

 

Conclusion:

With S9 metabolic activation:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment with test chemical. The results showed evidence of gene toxicity when cells were exposed to test chemical at 10 mM, while treatment with 5 mM or less did not cause gene toxicity. Therefore, it is considered that test chemical in the concentrations of 5 mM or below does not cause genetic mutation(s), while concentrations at 10 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation.

 

Without S9 metabolic activation:

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and without metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment with the test chemical. Independently of treatment concentration of test chemical, the results showed no evidence of potential gene toxicity. Therefore, it is considered that test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation.

 

In another study the test chemical was tested for mutagenicity in L5178Y mouse lymphoma cells. In the study, the test chemical was tested with and without metabolic activation up to cytotoxic doses.Test chemical was concluded to be non-mutagenic in mouse lymphoma cells.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in mammalian cell line in the presence and absence of metabolic activation 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 weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and In vitro gene mutation study in mammalian cells. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.