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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

Zinc Distearate was observed for its mutagenic potential in Salmonella typhimurium TA 1535, TA 1537, TA 1538 ,TA 98 and TA 100 by plate assay and suspension tests in the presence and absence of S9. The test result was considered to be non mutagenic in both the test.

In vitro mammalian chromosome aberration study:

The test chemical does not induce chromosome aberration in mammalian 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.

In vitro gene mutation study in mammalian cells:

When CHO cells were exposed to test chemical in the concentration of 0, 1, 2.5 or 5 mM the results did not show any evidence of gene toxicity, whereas a concentration of 10 mM showed evidence of gene toxicity in the presence of metabolic activation. The test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM showed no evidence of potential gene toxicity in CHO cells when exposed to the test chemical in the absence of metabolic activation..

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from NTRL
Qualifier:
according to
Guideline:
other: As mention below
Principles of method if other than guideline:
The objective of this study was to evaluate-the test compaund) for genetic activity in microbial assays with and without the addition of mammalian metabolic activation preparations.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
other: TA 1535, TA 1537, TA 1538 ,TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not specified
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
The tissue homogenates and 9,000 x g supernatants were prepared from tissues of the following mammalian species: Mouse - ICR random bred adult males; at - Sprague-Dawley adult males; and monkey - Macaca mulatta adult males
Test concentrations with justification for top dose:
O. 115, 0.0575 and 0.02875%
Vehicle / solvent:
Vehicle
- Vehicle(s)/solvent(s) used: N, N-Dimethyl Formamide
- Justification for choice of solvent/vehicle: Test chemical was soluble in DMF
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
N, N-Dimethyl Formamide
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9;Methylnitrosoguanidine Ethylmethanesulfonate 2-Nitrofluorene Quinacrine mustard +S9; Dimethylnitrosamine 2-Acetylaminofluorene 8-Aminoquinoline 2-Aminoanthracene
Details on test system and experimental conditions:
Details on test system and conditions
METHOD OF APPLICATION;

Experiment I; Plate Tests(overlay method)
Experiment II; Suspension Tests

DURATION
Experiment I;
- Exposure duration:48 to 72 hours

Experiment II;
- Preincubation period: 1 hour
- Exposure duration:48 hours


DETERMINATION OF CYTOTOXICITY
- Method; relative total growth
Evaluation criteria:
Evaluation Criteria for Ames Assay
Because the procedures used to evaluate the mutagenicity of the test chemicalwere semi quantitative. the criteria used to determine positive effects are
inherently subjective and are based primarily on a historical data base. Most data sets are evaluated using the following criteria:
1. Strains TA-1535. TA-1537. and TA-1538
If the solvent control value is within the normal range. a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic.
2. Strains TA-98. TA-100. and 04
If the solvent control value is within the normal range. a chemical that produces a positive dose response over three concentrations with the highest increase equal to twice the solvent control value for TA-100 and two to three times the solvent control value for strains TA-98 and 04 is considered to be mutagenic. For these strains. the dose response increase should start at approximately the solvent control value.
3. Pattern
Because TA-1535 and TA-100 were both derived from the same parental strain (G46) and because TA-l~38 and TA-98 were both derived from the same parental strain (03052). There is a built-in redundancy in the microbial assay. In general the two strains of a set respond to the same mutagen and such a pattern is sought. It is also anticipated that if a given strain, e.g. TA1537,responds to a mutagen in nonactivation tests it will generally do so in activation tests. (The converse of this relationship is not expected.) While similar response patterns are not required for all mutagens. they can be used to enhance the reliability of an evaluation decision.
Statistics:
Yes
Key result
Species / strain:
other: Salmonella typhimurium TA 1535, TA 1537, TA 1538 ,TA 98 and TA 100
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
Remarks on result:
other: No mutagenic effects were observed
Conclusions:
Zinc Distearate was observed for its mutagenic potential in Salmonella typhimurium TA 1535, TA 1537, TA 1538 ,TA 98 and TA 100 by plate assay and suspension tests in the presence and absence of S9. The test result was considered to be non mutagenic in both the test.
Executive summary:

In genetic toxicity study Zinc Distearate was assessed for its possible mutagenic potential by In vitro bacterial gene mutation assay. The test was performed inSalmonella typhimurium TA 1535, TA 1537, TA 1538 ,TA 98 and TA 100 by plate assay and suspension tests in the presence and absence of S9 . The test substance was exposed at the concentration of O. 115, 0.0575 and 0.02875%.No mutagenic effects were observed in both the assayin the presence and absence of S9.Therefore test substance was considered to be non-mutagenic with and without metabolic activation inSalmonella typhimurium TA 1535, TA 1537, TA 1538 ,TA 98 and TA 100.Hence the substance cannot be classified as gene mutant in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
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 from various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
1
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:
in vitro mammalian chromosome aberration test
Target gene:
Not specified
Species / strain / cell type:
other: Chinese hamster lung(CHL)cells
Remarks:
1
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Remarks:
2
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
1. -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

2. + S9 mix: Negative / Positive control, 0.5, 1.0 and 2.0 μg/mL
- S9 mix: Negative / Positive control, 0.5, 1.0 and 2.0 μg/mL
Vehicle / solvent:
1. - Vehicle(s)/solvent(s) used: 1% Carboxymethylcellulose sodium
2. - Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: The solubility test showed that the test substance was insoluble in water, dimethyl sulfoxide (DMSO), glycerol formal, but suspended in tetrahydrofuran. Tetrahydrofuran was selected as a vehicle.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
- Vehicle(s)/solvent(s) used: 1% Carboxymethylcellulose sodium
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9 mix, Mitomycin C +S9 mix, Cyclophosphamide
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
1% of tetrahydrofuran
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
2
Details on test system and experimental conditions:
1. Chinese hamster lung(CHL)cells were used .
2. - Short term exposure: CHL cells (5×10^4 cells per 5 mL in 60 mm petri-dish) were cultured for 72 hours and thereafter exposed in duplicate to the selected three doses of the test substance for 6 hours in the absence or in the presence of
S9 mix and recovered in fresh medium for 18 hours.
- Continuous exposure: CHL cells (5×10^4 cells per 5 mL in 60 mm petri-dish) were cultured for 72 hours and thereafter exposed in duplicate to the selected three doses of the test substance for 24 hours in the absence of S9 mix.
- Chromosome preparation: Cell cultures were treated with Colcemid for 2 hours prior to harvesting. Thereafter the cell cultures were centrifuged for 5 minutes at 1200 rpm and the supernatant was removed. Cells in the remaining pellet were swollen by a 30 min treatment with hypotonic solution (0.075 M KCl pre-warmed at 37􀬇). After hypotonic treatment, cells were fixed with 3 times of methanol: glacial acetic acid fixative (3 : 1 v/v).
- Slide preparation: Fixed cells were dropped onto pre-cleaned microscope slides which were then allowed to air-dry and thereafter stained in 5% (v/v) Giemsa solution.
- Slide analysis: All slides, including those of positive and negative controls, were independently coded before microscopic analysis. At least 200 well-spread metaphases were scored per concentration and control.
Rationale for test conditions:
No data
Evaluation criteria:
1. The cells were observed for chromosomal abbreviation, gaps.
2. 1) In each concentration, the number of aberrant cells in 200 metaphase cells was presented. Structural aberration was evaluated by classifying to chromosomal
2) Break and exchange, and chromatid break and exchange. A gap was defined as the deletion smaller than the width of chromatid and enumerated, nonetheless, it was not included in the total aberration frequency. As numerical aberration, only polyploidy higher than tetraploid.
3) endoreduplication were evaluated. A cell with more than one type of structural aberration was counted as
4) one positive cell, their percentage of aberration cell was estimated and considered to be the frequency of chromosome aberration. In addition, chromosome aberration types were recorded separately.
Statistics:
2. 1) Statistical analysis of chromosomal aberration frequency excluding gaps was performed according to the OECD guideline.
2) In 200 metaphase cells of each group, the numbers of aberrant cells and normal cells were presented.
3) If the dose-dependent increase or the repeated increase was detected. It was evaluated to be positive. The significant difference in the frequence of chromosomal aberration
4) between the negative control and the test substance treatment group was analyzed using Fisher's exact test (Altman, 1993). The test substance was considered to be negative if P-value was lower than 0.05.
Species / strain:
other: CHL/IU cells
Remarks:
1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
1. 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 effect were observed
Conclusions:
The test chemical does not induce chromosome aberration in mammalian 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.
Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Genetic toxicity in vitro study was 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 -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, gaps were observed in 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.

Chromosome aberration study was performed to evaluate the mutagenic nature of the test chemical. The study was performed using cultured Chinese hamster lung fibroblast (CHL cells) at seven dose levels, four soluble concentrations 0.13, 0.25, 0.5, 1.0 ug/mL and three insoluble concentrations 5, 10, 20 ug/mL in the presence and absence of a metabolic activation system (rat liver S9 mix).CHL cells were treated with the test substance for 6 hours, changed to fresh culture medium, and cultured for total 24 hours. In the presence of S9 mix, the frequency of structural chromosome aberrations was not significantly induced at the three concentrations, 0.5, 1.0, 2.0 ug/mL, and similarly, in the absence of S9 mix, the frequency of aberrant cells was not induced at the three concentrations, 0.5, 1.0, 2.0 ug/mL (p>0.05, each). In addition, treating with the test substance at 0.5, 1.0, 2.0 ug/mL for 24 hours in the absence of S9 mix, a significant induction was not detected in regard to the frequency of aberrant cells (p>0.05). The frequency of chromosome aberration by the vehicle control was found to be within a normal range for the acceptability of the test, the positive control substances, cyclophosphamide (with S9 mix) and mitomycin C (without S9 mix) induced statistically significant chromosomal aberrations under each condition (p<0.001 each) and hence is not likely to classify as a gene mutant in vitro. Based on the observations made, the test chemical did not showe any evidence of clastogenic activity in mammalian cell culture system in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical does not induce chromosome aberration in mammalian 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The purpose of this study was to assess toxic and genotoxic effects of test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
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.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
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
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
0, 1, 2.5, 5 or 10 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/ vehicle: Test chemical was dissolved in ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: N-ethyl-N-nitrosourea (ENU) (Without S9)
Details on test system and experimental conditions:
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
Rationale for test conditions:
No data
Evaluation criteria:
Dose dependent increase in the gene mutation activity
Statistics:
No data
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
At 10 mM
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No data

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

2/4a

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/Aa

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:
When CHO cells were exposed to test chemical in the concentration of 0, 1, 2.5 or 5 mM the results did not show any evidence of gene toxicity, whereas a concentration of 10 mM showed evidence of gene toxicity in the presence of metabolic activation.

test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM showed no evidence of potential gene toxicity in CHO cells when exposed to the test chemical in the absence of metabolic activation..
Executive summary:

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The data available for the test chemical and its various read across chemicals was reviewed to determine the mutagenic nature of Zinc Distearate (ZDS) (CAS no. 557-05-1). The studies are as mentioned below:

AMES assay:

In genetic toxicity study Zinc Distearate was assessed for its possible mutagenic potential by In vitro bacterial gene mutation assay. The test was performed inSalmonella typhimurium TA 1535, TA 1537, TA 1538 ,TA 98 and TA 100 by plate assay and suspension tests in the presence and absence of S9 . The test substance was exposed at the concentration ofO. 115, 0.0575 and 0.02875%. No mutagenic effects were observed in both the assayin the presence and absence of S9.Therefore test substance was considered to be non-mutagenic with and without metabolic activation inSalmonella typhimurium TA 1535, TA 1537, TA 1538 ,TA 98 and TA 100.Hence the substance cannot be classified as gene mutant in vitro.

This is further supported by another study. 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 test chemical using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay. The test compound was used at a dosage level of 0, 1, 3, 10, 33, 100, 333, 166 µg/plate in the preincubation assay of 48 hrs. Test chemical failed to induce mutation in the S. typhimurium tester strains TA1535, TA97, TA98 and TA100 and hence is negative for mutation in vitro.

Genetic toxicity in vitro study was also assessed for test chemical. For this purpose AMES test was performed according to OECD 471,472and 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. 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.

In vitro mammalian chromosome aberration study:

Genetic toxicity in vitro study was 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 -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, gaps were observed in 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.

Chromosome aberration study was performed to evaluate the mutagenic nature of the test chemical. The study was performed using cultured Chinese hamster lung fibroblast (CHL cells) at seven dose levels, four soluble concentrations 0.13, 0.25, 0.5, 1.0 ug/mL and three insoluble concentrations 5, 10, 20 ug/mL in the presence and absence of a metabolic activation system (rat liver S9 mix).CHL cells were treated with the test substance for 6 hours, changed to fresh culture medium, and cultured for total 24 hours. In the presence of S9 mix, the frequency of structural chromosome aberrations was not significantly induced at the three concentrations, 0.5, 1.0, 2.0 ug/mL, and similarly, in the absence of S9 mix, the frequency of aberrant cells was not induced at the three concentrations, 0.5, 1.0, 2.0 ug/mL (p>0.05, each). In addition, treating with the test substance at 0.5, 1.0, 2.0 ug/mL for 24 hours in the absence of S9 mix, a significant induction was not detected in regard to the frequency of aberrant cells (p>0.05). The frequency of chromosome aberration by the vehicle control was found to be within a normal range for the acceptability of the test, the positive control substances, cyclophosphamide (with S9 mix) and mitomycin C (without S9 mix) induced statistically significant chromosomal aberrations under each condition (p<0.001 each) and hence is not likely to classify as a gene mutant in vitro. Based on the observations made, the test chemical did not showe any evidence of clastogenic activity in mammalian cell culture system in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical does not induce chromosome aberration in mammalian 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.

In vitro gene mutation study in mammalian cells:

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.

Based on the data available for the target chemical and its read across chemicals and applying the weight of evidence approach, Zinc Distearate (ZDS) (CAS no 557-05-1) does not exhibit mutagenic activity. Hence the test substance can not be classified as gene mutant in vitro. Hence is not likely to classify as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical and its read across chemicals and applying the weight of evidence approach, Zinc Distearate (ZDS) (CAS no 557-05-1) does not exhibit mutagenic activity. Hence the test substance can not be classified as gene mutant in vitro. Hence is not likely to classify as per the criteria mentioned in CLP regulation.