Sorbitan monostearate, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in distilled water or DMSO as the solvent and used at dose level of 0, 10, 100, 200, 1000 or 2000 µg/plate. The exposure duration was 48 hrs at 37˚C. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Chromosome aberration assay:

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. The study was performed using Chinese hamster fibroblast cell line (CHL) cell line. The test chemical was dissolved in physiological saline and used at dose level of 0.20 mg/mL. Three different doses, including the 50% inhibition dose of each agent, which was estimated by the growth inhibition test described above, were prepared and separately added to 3-day-old cultures (about 10⁵cells/6-cm dish). Chromosome preparations were made, as a rule at 24 and 48 h, or when necessary at 6 h, after the treatment. Cells were treated with colcemid (0.2µg/ml) for 2 h, and after trypsinization, they were incubated in 0.075 M KC1 hypotonic solution for 15 rain at 37°C. The cells were fixed with ice-cold fixative (methanol : glacial acetic acid, 3 : 1 v/v) which was changed 3 times. A few drops of the suspension were then placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1% Giemsa's buffered solution (pH 6.8) for 20 min. The number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases at the magnification of 700. Types of aberration were classified into 5 groups: chromatid gaps (g), chromatid breaks (b), chromatid or chromosomal translocation (t), ring formation (r) and fragmentation or pulverization (f). Breaks less than the width of a sister chromatid were designated as gaps in our criteria. The incidence of polyploid cells was also calculated. Untreated cells and cells treated only with solvent served as controls. Based on the observations made, the test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line (CHL) and hence is 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:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

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:

S9 metabolic activation system

Test concentrations with justification for top dose:

0, 10, 100, 200, 1000 or 2000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

other: N-Methyl-N’-nitro-N-nitrosoguanidine, 2-Acetylaminofluorene, N-Nitrosodimethylamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- 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: No data

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:

The plates were observed or a dose dependent increase in the number of revertants/plate

Statistics:

No data

Species / strain:

S. typhimurium, other: TA98 and TA100

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:

No data

Remarks on result:

other: No mutagenic potential

Table 1. Mutagenicity of the test compound and the respective control chemicals

Compound	Dose (µg/plate)	No. of His+ revertants/plate
		TA100		TA98
		-S9	+S9	-S9	+S9
Distilled water	-	145	137	19	32
DMSO	-	151	141	23	28
4-Nitroquinoline 1-oxide	0.5	1588	148	167	36
Benzo[a]pyrene	2	4152	128	27	30
2-Acetylaminofluorene	5	178	928	35	614
N-Nitrosodimethylamine	50	128	1080	18	2292
Test chemical	10	121	111	18	31
	100	134	127	23	29
	200	157	13	33	34
	1000	148	141	22	33
	2000	115	136	24	33

 

Conclusions:

The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98 and TA100 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 study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in distilled water or DMSO as the solvent and used at dose level of 0, 10, 100, 200, 1000 or 2000 µg/plate. The exposure duration was 48 hrs at 37˚C. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: Chinese hamster fibroblast cell line (CHL)

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells had been maintained by 5-day passages and grown in a monolayer in petri dishes with Eagle's MEM (GIBCO F-11) supplemented with 10% calf serum. Their doubling time was estimated as 18.2 h at their exponential growth at 37°C in a 5% CO2 atmosphere.
- Properly maintained: Yes, The cells had been maintained by 5-day passages
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

Maximum dose: 0.20 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: physiological saline (S)
- Justification for choice of solvent/vehicle: The test chemical was soluble in physiological saline

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Physiological saline

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium
Cells at the start of test: 105 cells/6-cm dish

DURATION
- Preincubation period: No data
- 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): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: The number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases at the magnification of 700.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, Aliquots were removed for determination of cell count and viability assessment via the trypan blue exclusion
Test. Mitotic index was also determined

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

OTHER: The cells were fixed with ice-cold fixative (methanol : glacial acetic acid, 3 : 1 v/v) which was changed 3 times. A few drops of the suspension were then placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1% Giemsa's buffered solution (pH 6.8) for 20 min.

Rationale for test conditions:

No data

Evaluation criteria:

Types of aberration were classified into 5 groups: chromatid gaps (g), chromatid breaks (b), chromatid or chromosomal translocation (t), ring formation (r) and fragmentation or pulverization (f). Breaks less than the width of a sister chromatid were designated as gaps

CHL cells commonly have less than 3.0% cells with chromosomal aberrations. Therefore, the final judgement given to all experimental groups was asfollows. Negative (--) if less than 4.9% of the aberration was detected even when doses of the agent were elevated to sub-lethal amounts, where almost nomitosis was observed; suspicious (+) if between 5.0 and 9.9%, and positive if between 10.0 and 19.9% (+), 20.0 and 49.9% (++) or more than 50.0% (+++). When no reasonable dose response was obtained, additional experiments with different doses were carried out to confirm its reproducibility

Statistics:

No data

Species / strain:

mammalian cell line, other: Chinese hamster fibroblast cell line (CHL)

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:

No data

Remarks on result:

other: No mutagenic potential

Table: Summary of chromosome aberration assay results

Chemical	Solvent	Maximum dose (mg/mL)	Chromosomal aberration test
			%	Time	Type	Judge
Test chemical	Physiological saline	0.20	1.0	48	Chromatid breaks	-

Conclusions:

The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line (CHL) and hence is it is not likely to classify as a gene mutant in vitro.

Executive summary:

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. The study was performed using Chinese hamster fibroblast cell line (CHL) cell line. The test chemical was dissolved in physiological saline and used at dose level of 0.20 mg/mL. Three different doses, including the 50% inhibition dose of each agent, which was estimated by the growth inhibition test described above, were prepared and separately added to 3-day-old cultures (about 10⁵cells/6-cm dish). Chromosome preparations were made, as a rule at 24 and 48 h, or when necessary at 6 h, after the treatment. Cells were treated with colcemid (0.2µg/ml) for 2 h, and after trypsinization, they were incubated in 0.075 M KC1 hypotonic solution for 15 rain at 37°C. The cells were fixed with ice-cold fixative (methanol : glacial acetic acid, 3 : 1 v/v) which was changed 3 times. A few drops of the suspension were then placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1% Giemsa's buffered solution (pH 6.8) for 20 min. The number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases at the magnification of 700. Types of aberration were classified into 5 groups: chromatid gaps (g), chromatid breaks (b), chromatid or chromosomal translocation (t), ring formation (r) and fragmentation or pulverization (f). Breaks less than the width of a sister chromatid were designated as gaps in our criteria. The incidence of polyploid cells was also calculated. Untreated cells and cells treated only with solvent served as controls. Based on the observations made, the test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line (CHL) and hence is 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 target chemical was reviewed to determine the mutagenic nature of Sorbitan monostearate, ethoxylated (CAS no 9005 -67 -8). 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 using Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in distilled water or DMSO as the solvent and used at dose level of 0, 10, 100, 200, 1000 or 2000 µg/plate. The exposure duration was 48 hrs at 37˚C. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98 and TA100 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 mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. The study was performed using Chinese hamster fibroblast cell line (CHL) cell line. The test chemical was dissolved in physiological saline and used at dose level of 0.20 mg/mL. Three different doses, including the 50% inhibition dose of each agent, which was estimated by the growth inhibition test described above, were prepared and separately added to 3-day-old cultures (about 10⁵cells/6-cm dish). Chromosome preparations were made, as a rule at 24 and 48 h, or when necessary at 6 h, after the treatment. Cells were treated with colcemid (0.2µg/ml) for 2 h, and after trypsinization, they were incubated in 0.075 M KC1 hypotonic solution for 15 rain at 37°C. The cells were fixed with ice-cold fixative (methanol : glacial acetic acid, 3 : 1 v/v) which was changed 3 times. A few drops of the suspension were then placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1% Giemsa's buffered solution (pH 6.8) for 20 min. The number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases at the magnification of 700. Types of aberration were classified into 5 groups: chromatid gaps (g), chromatid breaks (b), chromatid or chromosomal translocation (t), ring formation (r) and fragmentation or pulverization (f). Breaks less than the width of a sister chromatid were designated as gaps in our criteria. The incidence of polyploid cells was also calculated. Untreated cells and cells treated only with solvent served as controls. Based on the observations made, the test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line (CHL) and hence is it is not likely to classify as a gene mutant in vitro.

The negative results for bacterial reverse mutation assay and chromosomal aberration assay is further suppoerted by the data from mammalian cell transformation assay study.

In vitro mammalian cell transformation assay was performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in DMSO as the solvent and used at dose level of 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20, 50, 100 or 300 µg/mL. Pregnant Syrian golden hamsters were killed on days 13 and 14 of gestation for preparation of target cells and feeder-layer cells respectively. Embryos without heads and viscera were minced with scissors, and trypsinized with 0.25% trypsin. Inocula of 10000000 embryo cells per 75-cm’ flask were incubated at 37°C in a humidified atmosphere of 10% CO2 in air. When they became confluent primary cultures were trypsinized, dispensed in lots of 5000000 cells in glass ampules, and stored in liquid nitrogen for use as target and feeder-layer cells. The assay takes 15 days from start to finish. On Day 0, an ampule of cryopreserved primary cells prepared as feeder-layer cells was rapidly thawed and plated in a 75-cm²flask containing 20 ml of the culture medium. The medium was changed every day. On day 3, an ampule of cryopreserved primary cells prepared as target cells was also rapidly thawed and plated in a 75-cm²flask. On day 4, the feeder cells which were shifting from a stage of logarithmic growth to a stationary phase were irradiated with 5000 R from a linear accelerator, trypsinized, and then plated at 6 x 10⁴tells/60-mm dish in 2 ml of complete medium. On day 5, the target cells which were approximately 80 -90% confluent were trypsinized, and a suspension of 500 target cells in 2 ml of complete medium was then added to each of the dishes plated the day before with irradiated feeder-layer cells. On day 6, an appropriate dose of the test chemical in a volume of 4 ml was added, giving a total volume of 8 ml of medium in each dish. Nine dishes were used for each dose level in all the experiments (in a few cases only eight or seven dishes were used for controls). On day 14, the cultures were fixed with absolute methanol for 10min and stained with Giemsa solution for 45 min or more. The stained dishes were examined with a stereoscopic dissection microscope to count normal and transformed colonies. The exposure duration was 8 days. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce cell transformation in the Pregnant Syrian golden hamsters embryo cell line used and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was perfomed using Salmonella typhimurium strains TA98 and TA 100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved DMSO as the solvent and used at dose level of 0, 10, 100 or 1000µg/plate. The exposure duration was 48 hrs at 37˚C. Concurrent solvent and positive control chemicals were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Rec assay was also performed to determine the mutagenic nature of the test chemical. The study was performed using Bacillus subtilis H17 and M45. The test chemical was dissolved in DMSO and used at dose level of 0, 0.05, 0.5 or 5.0 mg/disk. The disk was 8 mm in diameter. The strain was placed so as to cover the starting point of the strain, cultured overnight at 37 ° C. The length of growth inhibition zone of both strains was measured. Concurrent solvent and negative control plates were also included in the study. The test chemical did not induce growth inhibition in Bacillus subtilis H17 and M45 and hence it is not likely to classify as a gene mutant in vitro.

In another study, Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The test chemical was dissolved in distilled water or DMSO as the solvent and used at dose level of 0, 10, 100, 200, 1000 or 5000 µg/plate in experiment 1, 2 and 3. Concurrent solvent and negative control chemicals were also included in the study. The test chemical did not induce gene mutation in Salmonella typhimurium strains TA98 and TA100 in the presence and absence of rat, hamster and guinea pig isolated S9 metabolic activation system and also in the presence and absence of norharman and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the test chemical, Sorbitan monostearate, ethoxylated (CAS no 9005 -67 -8) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro. The negative results of the bacterial reverse mutation assay and chromosomal aberration study are also supported by the the negative results of the in vitro mammalian cell transformation assay indicating the non-mutagenic nature of the test chemical as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the test chemical, Sorbitan monostearate, ethoxylated (CAS no 9005 -67 -8) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro. The negative results of the bacterial reverse mutation assay and chromosomal aberration study are also supported by the the negative results of the in vitro mammalian cell transformation assay indicating the non-mutagenic nature of the test chemical as per the criteria mentioned in CLP regulation.