Sorbitan, tridocosanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA pKM101, with and without metabolic activation

 

Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): negative in Chinese Hamster Lung (CHL/IU) cells, with and without metabolic activation

 

Gene mutation in mammalian cells (OECD 476, HPRT test): negative in Chinese Hamster Ovary (CHO) cells, with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Dec 2020 - 09 Mar 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted in 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Ogyéi National Institute of Pharmacy and Nutrition, Budapest, Hungary

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

Subline K1

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: American Type Culture Collection (ATCC, Manassas, Virginia, United States)
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: Cells were cultivated in F12-10 culture medium (see below) and incubated at 37 ± 0.5 °C in a humidified atmosphere at 5 ± 0.3° CO2 in air.
- Periodically ‘cleansed’ of spontaneous mutants: yes. Prior to use in the test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium.

MEDIA USED
- F12-1 medium: Treatment medium for 5 h treatment -S9 mix
Ham's F12 medium supplemented with 1% heat inactivated foetal bovine serum (FBS), 0.001 mL/mL L-glutamine and 0.01 mL/mL antibiotic/antimycotic solution (consisting of 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B)

- F12-5 medium: Treatment medium for 5 h treatment +S9 mix
Ham's F12 medium supplemented with 5% heat inactivated FBS, 0.001 mL/mL L-glutamine and 0.01 mL/mL antibiotic/antimycotic solution (consisting of 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B)

- F12-10 medium: General culture medium
Ham's F12 medium supplemented with 10% heat inactivated FBS, 0.001 mL/mL L-glutamine and 0.01 mL/mL antibiotic/antimycotic solution (consisting of 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B)

- F12-SEL medium: Culture medium for selection
Hypoxanthine-free Ham's F12 medium supplemented with 10% heat inactivated FBS, 0.001 mL/mL L-glutamine and 0.01 mL/mL antibiotic/antimycotic solution (consisting of 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Cofactor supplemented post mitochondrial fraction (S9 mix).
- source of S9: in house of the testing facility
- method of preparation of S9 mix: S9 mix was prepared from the livers of male Wistar rats (502 - 672 g) that were 5 months old. The animals were induced with phenobarbital and β-naphthoflavone at 80 mg/kg bw/day by oral gavage for three consecutive days. Liver homogenates were prepared 4 days after induction.
S9 mix was composed of 20 mM HEPES, 330 mM KCl, 50 mM MgCl2, 40 mM NADP, 50 mM D-glucose-6-phosphate and S9 fraction (30% in S9 mix).
- concentration or volume of S9 mix in the final culture medium: 10%
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The biological activity of the S9 mix was demonstrated in an Ames test using the reference mutagens 2-aminoanthracene and benzo[a]pyrene.

Test concentrations with justification for top dose:

Preliminary cytotoxicity test:
With and without metabolic activation: 3.906, 7.813, 15.625, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL (5 h)

Experiment 1:
With and without metabolic activation: 2.74, 8.23, 24.69, 74.07, 222.22, 666.67 and 2000 µg/mL (5 h)

Experiment 2:
With metabolic activation: 2.74, 8.23, 24.69, 74.07, 222.22, 666.67 and 2000 µg/mL (5 h)
Without metabolic activation: 2.74, 8.23, 24.69, 74.07, 222.22, 666.67 and 2000 µg/mL (24 h)

Justification for top concentration: Concentrations were selected based on the results of a preliminary cytotoxicity test. There was no cytotoxicity observed up to and including the maximum dose of 2000 µg/mL.

Vehicle / solvent:

- Vehicle/solvent used: Dimethyl sulfoxide (DMSO), 1% in the final culture medium

- Justification for choice of solvent/vehicle: In a trial experiment, the test item turned out to be insoluble in water. Formulations in DMSO and in 1% aqueous methyl cellulose solution gave visibly homogeneous suspensions. Therefore, DMSO was selected as vehicle of the study.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2 x 10E6 (in dishes of 100 mm diameter)
- Test substance added in medium (F12-1 for 5 h exposure ± S9 mix, F12-5 for 24 h exposure –S9 mix).

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 h with and without S9 mix (Experiments 1 and 2) and 24 h without S9 mix (Experiment 2)
- Expression time: At the end of the treatment period, the cultures were re-plated and incubated for a 7 days expression period. The cells were sub-cultured and maintained at 2 x 10E5 cells/dish twice.
- Selection time: After the expression period, 4 x 10E5 cells were plated in 100 mm dishes (5 per sample) for determination of mutant frequency and incubated for 7 days.
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days

SELECTION:
- selection agent: 20 µg/mL 6-thioguanine (6-TG) was added to the medium for the 7-days incubation period of mutant selection.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival (RS) and cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY:
The mutant frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10E6 cells: 5 plates at 4x10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and were expressed as 6-TG resistant mutants per 10E6 clonable cells.

Evaluation criteria:

The test item was considered to be mutagenic in this assay if the following criteria were met:
1. The assay is valid.
2. The mutant frequency at one or more doses is significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency is reproducible.
4. There is a dose-response relationship.
5. The historical control range is considered when deciding if the result is positive.
Results which only partially met the criteria were dealt with on a case-by-case basis (historical control data of untreated control samples was taken into consideration if necessary). According to the relevant OECD 476 guideline, the biological relevance of the results was considered first, statistical significance was not the only determination factor for a positive response.

Statistics:

The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the nonparametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using MannWhitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2010 software (R-squared values were calculated for the log concentration versus the mutation frequency). In the statistical analysis, negative trends were not considered significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

In Exp. 2 (5 h exposure +S9 mix) a statistically significant increase in mutant frequency was observed at 24.69 µg/mL. The effect was not dose dependent and within historical control data and therefore considered not relevant.

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There were no significant changes in pH after treatment.
- Data on osmolality: There were no significant changes in osmolality after treatment.
- Precipitation and time of the determination: Precipitation was observed in both experiments at 24.69 – 2000 µg/mL at the end of treatment in the presence and absence of metabolic activation. The precipitate did not interfere with the reading of the results.

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed using test item concentrations in the range of 3.906 – 2000 µg/mL in the presence and absence of metabolic activation. The cells were exposed for 5 h ±S9 mix and for 24 h –S9 mix. There was no cytotoxicity observed at any concentration, neither with nor without S9 mix. Precipitation was observed at 15.625 – 2000 µg/mL after 5 h exposure with S9 mix and at 31.25 – 2000 µg/mL after 5 or 24 h without S9 mix.

STUDY RESULTS
Cytotoxicity:
There was no cytotoxicity observed in any experiment up to the highest dose tested, neither in presence nor absence of metabolic activation.

Mutagenicity:
In the first experiment, no statistically significant increase in the mutant frequency was observed at any dose level, neither with nor without S9 mix. In the second experiment after 5 h exposure with S9 mix, a statistically significant increase in mutant frequency was observed at 24.69 µg/mL. The finding was not dose-related and fell within the range of the laboratory’s historical control data and was therefore not considered relevant. The test item was concluded to be negative for mutagenicity in mammalian cells in vitro. For details on experimental results, please refer to Tables 1 and 2 under “Any other information on results incl. tables”.
- Concurrent vehicle, negative and positive control data were in accordance with the general historical control data generated in the testing facility for all assays. The positive controls DMBA in the presence of S9 mix and EMS in the absence of S9 mix induced statistically significant increases in the mutant frequency over those of controls, thus confirming the functionality of the S9 mix and demonstrating the sensitivity of the test.

HISTORICAL CONTROL DATA: Please refer to Table 3 under “Any other information on results incl. tables”.

Table 1: Results of Experiment 1

S9	Treatment	Concentration	Survival			Viability		Mutagenicity
			Total no. of colonies	Cloning efficiency (CE)	Relative survival (%)	Total no. of colonies	Cloning efficiency (CE)	Total no. of colonies	Mutant frequency
+	5	2000 µg/mL (P)	1179	0.983	89	1123	0.936	24	6.4
		666.67 µg/mL (P)	1177	0.981	89	978	0.815	19	5.8
		222.22 µg/mL (P)	1202	1.002	91	1142	0.952	18	4.7
		74.07 µg/mL (P)	1089	0.908	83	1117	0.931	25	6.7
		24.69 µg/mL (P)	1374	1.145	104	1162	0.968	26	6.7
		8.23 µg/mL	1260	1.050	96	1157	0.964	22	5.7
		2.74 µg/mL	1219	1.016	92	1121	0.934	21	5.6
		Negative control	1319	1.099	100	1141	0.951	22	5.8
		Untreated control	1196	0.997	91	1092	0.910	23	6.3
		Positive control (DMBA)	59	0.049	4	1188	0.990	2244	566.3**
-	5	2000 µg/mL (P)	1152	0.960	107	1126	0.938	21	5.7
		666.67 µg/mL (P)	1172	0.977	109	1181	0.984	32	8
		222.22 µg/mL (P)	1321	1.101	123	1077	0.898	21	5.8
		74.07 µg/mL (P)	1284	1.070	120	1047	0.873	19	5.4
		24.69 µg/mL (P)	1306	1.088	122	1248	1.040	22	5.4
		8.23 µg/mL	1203	1.003	112	1122	0.935	26	7
		2.74 µg/mL	1189	0.991	111	1020	0.850	25	7.3
		Negative control	1074	0.895	100	1185	0.988	26	6.6
		Untreated control	1234	1.028	115	1180	0.983	20	5.1
		Positive control (EMS)	899	0.749	84	970	0.808	1089	337.0**
DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL; EMS = Ethyl methanesulfonate, 0.4 µL/mL; (P): precipitation observed at the end of treatment

Table 2: Results of Experiment 2

S9	Treatment	Concentration	Survival			Viability		Mutagenicity
			Total no. of colonies	Cloning efficiency (CE)	Relative survival (%)	Total no. of colonies	Cloning efficiency (CE)	Total no. of colonies	Mutant frequency
+	5	2000 µg/mL (P)	1475	1.229	122	1127	0.939	22	5.9
		666.67 µg/mL (P)	1235	1.029	102	1051	0.876	23	6.6
		222.22 µg/mL (P)	1290	1.075	106	1060	0.883	32	9.1
		74.07 µg/mL (P)	1232	1.027	102	1113	0.928	21	5.7
		24.69 µg/mL (P)	1264	1.053	104	1048	0.873	35	10.1*
		8.23 µg/mL	1152	0.960	95	1003	0.836	37	11
		2.74 µg/mL	1194	0.995	98	1090	0.908	42	11.4
		Negative control	1213	1.011	100	1125	0.938	29	7.7
		Untreated control	1179	0.983	97	1077	0.898	34	9.5
		Positive control (DMBA)	95	0.079	8	1115	0.929	1687	453.3**
-	24	2000 µg/mL (P)	1267	1.056	97	1179	0.983	24	6.1
		666.67 µg/mL (P)	1361	1.134	104	1042	0.868	20	5.7
		222.22 µg/mL (P)	1406	1.172	108	1144	0.953	23	6
		74.07 µg/mL (P)	1332	1.110	102	1104	0.920	22	6
		24.69 µg/mL (P)	1256	1.047	96	1129	0.941	38	10
		8.23 µg/mL	1344	1.120	103	1130	0.942	21	5.6
		2.74 µg/mL	1258	1.048	96	1016	0.847	28	8.3
		Negative control	1304	1.087	100	1137	0.948	30	8
		Untreated control	1223	1.019	94	1213	1.011	37	9.2
		Positive control (EMS)	768	0.640	59	472	0.393	1120	710.4**
DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL; EMS = Ethyl methanesulfonate, 0.4 µL/mL; (P): precipitation observed at the end of treatment

 

 

Table 3: Historical control data generated in the testing facility (updated in Oct 2017 using data of GLP studies)

	Mutation frequency (Number of 6-TG resistant mutants per 10E6 clonable cells)
	Untreated control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	18.3	20.7	19
standard deviation	15.1	16.4	17.2
minimum	5.1	5.5	3.3
maximum	64.1	55.5	58
n	27	13	14
	DMSO control
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	21.8	18.9	18.4
standard deviation	15.9	11.6	14.4
minimum	5.4	6.5	6.8
maximum	57.3	47.4	48.5
n	29	13	14
	Distilled water / Water based vehicle control
mean	11.5	9.1	15.5
standard deviation	3.8	3.4	5.6
minimum	6.1	5.2	9.2
maximum	15.8	11.6	20.1
n	6	3	3
	Positive controls
	DMBA	EMS	EMS
	5-hour, S9+	5-hour, S9-	24-hour, S9-
mean	905.2	445.6	1176.6
standard deviation	562.7	118.6	610.9
minimum	141.2	239.6	363.1
maximum	2119.4	636.6	2449.8
n	27	13	14

 

Conclusions:

Interpretation of results: negative in CHO cells with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 - 29 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon, trp operon

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Following concentrations were used in the two main experiments (preincubation), based on the results of the range finding study (preincubation):
All strains: 313, 625, 1250, 2500 and 5000 μg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)
- Justification for choice of solvent/vehicle: A preliminary solubility test showed that it was not possible to dissolve or uniformly suspend the test substance in water, DMSO or acetone. Thus, THF was selected as solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: 2-nitrofluorene (2NF), 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation (first and second experiment)

DURATION
- Preincubation period: until solidification takes place
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates in two experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn and number of revertant colonies

Evaluation criteria:

Acceptance criteria
The study was considered valid if:
- mean number of revertant colonies of the negative (solvent) and positive controls are in the historical control range
- no contamination
- mean number of revertant colonies in the positive control group is increased at least twice compared to the negative control group

Evaluation criteria
The number of revertant colonies in any strains at one or more doses is increased at least two times compared to the negative control group. There should be dose dependency or reproducibility as dose increases.

Statistics:

Individual plates were counted for revertant colonies. The average and standard deviation of the number of revertant colonies were calculated. Statistical analysis was not performed.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was observed at a concentration of 313 μg/plate or more (+/- S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was observed at a concentration of 313 μg/plate or more (+/- S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was observed at a concentration of 313 μg/plate or more (+/- S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was observed at a concentration of 313 μg/plate or more (+/- S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was observed at a concentration of 313 μg/plate or more (+/- S9 mix).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A dose range finding study (preincubation) was conducted to determine the highest dose for the main study. The six tested concentrations were ranging from 4.88 until 5000 μg/plate. No signs of growth inhibition by the test substance was evident at any tested concentration in the absence or presence of metabolic activation. The test substance precipitated at a concentration of 19.5 μg/plate or more in all strains in the presence and absence of metabolic activation. However, it did not interfere with the colony counting.

HISTORICAL CONTROL DATA
see Table 3 in "Any other information on results incl. tables"

Table 1: Test results (experiment 1, preincubation)

With or without S9 Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA 98	TA 1537	TA 100	TA 1535	WP2 uvrA
–	Solvent control (MEK)	20 ± 1	9 ± 1	109 ± 3	14 ± 1	121 ± 4
	313	22 ± 2	9 ± 1	118 ± 9	13 ± 1	113 ± 4
	625	22 ± 2	I I ± 2	124 ± 2	17 ± 2	119 ± 5
	1250	20 ± 1	9 ± 1	124 ± 2	18 ± 3	114 ± 1
	2500	21 ± 2	8 ± 3	125 ± 5	17 ± 2	112 ± 2
	5000	19 ± 2	8 ± 1	110 ± 2	13 ± 2	112 ± 2
	Positive controls (µg/plate)	2NF (5)	9AA (80)	SA (1.5)	SA (1.5)	4NQO (0.1)
	Mean (No. of colonies/plate)	799 ± 13	704 ± 20	695 ± 24	591 ± 11	615 ± 11
+	Solvent control (MEK)	35 ± 1	21 ± 1	122 ± 2	14 ± 1	162 ± 3
	313	39 ± 1	22 ± 2	133 ± 5	14 ± 2	161 ± 4
	625	38 ± 1	19 ± 2	129 ± 9	15 ± 2	163 ± 4
	1250	34 ± 3	16 ± 3	120 ± 5	13 ± 1	157 ± 2
	2500	34 ± 4	16 ± 1	124 ± 9	12 ± 2	147 ± 2
	5000	31 ± 2	18 ± 1	118 ± 4	12 ± 1	152 ± 5
	Positive controls (µg/plate)	2AA (1)	2AA (3)	2AA (2)	2AA (3)	2AA (2)
	Mean (No. of colonies/plate)	249 ± 37	152 ± 9	574 ± 30	111 ± 20	404 ± 17

2AA = 2-aminoanthracene

2NF = 2-nitrofluorene

4NQO = 4-nitroquinoline N-oxide

9AA = 9-aminoacridine

SAZ = sodium azide

The test substance precipitated at a concentration of 313 μg/plate or more in all strains in the presence and absence of metabolic activation.

Table 2: Test results (experiment 2, preincubation)

With or without S9 Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA 98	TA 1537	TA 100	TA 1535	WP2 uvrA
–	Solvent control (MEK)	20 ± 2	9 ± 1	108 ± 2	14 ± 1	116 ± 2
	313	19 ± 1	8 ± 1	114 ± 6	14 ± 2	117 ± 2
	625	20 ± 2	7 ± 1	110 ± 3	13 ± 1	116 ± 1
	1250	19 ± 2	8 ± 2	103 ± 4	12 ± 1	120 ± 2
	2500	16 ± 1	8 ± 1	108 ± 2	13 ± 2	116 ± 2
	5000	31 ± 2	7 ± 1	103 ± 3	11 ± 1	111 ± 1
	Positive controls (µg/plate)	2NF (5)	9AA (80)	SA (1.5)	SA (1.5)	4NQO (0.1)
	Mean (No. of colonies/plate)	761 ± 8	730 ± 23	683 ± 24	625 ± 13	633 ± 9
+	Solvent control (MEK)	36 ± 2	22 ± 1	121 ± 2	13 ± 2	160 ± 1
	313	38 ± 2	20 ± 1	122 ± 2	10 ± 1	151 ± 2
	625	40 ± 3	19 ± 1	118 ± 1	10 ± 1	152 ± 3
	1250	38 ± 4	15 ± 2	118 ± 2	10 ± 1	149 ± 3
	2500	30 ± 2	16 ± 1	118 ± 6	10 ± 1	144 ± 2
	5000	29 ± 2	16 ± 1	116 ± 4	10 ± 2	143 ± 1
	Positive controls (µg/plate)	2AA (1)	2AA (3)	2AA (2)	2AA (3)	2AA (2)
	Mean (No. of colonies/plate)	252 ± 13	153 ± 7	569 ± 43	118 ± 12	418 ± 14

2AA = 2-aminoanthracene

2NF = 2-nitrofluorene

4NQO = 4-nitroquinoline N-oxide

9AA = 9-aminoacridine

SAZ = sodium azide

The test substance precipitated at a concentration of 313 μg/plate or more in all strains in the presence and absence of metabolic activation.

Table 3: Historical data (negative and positive controls)

Strain		TA 98		TA100		TA 1535		TA 1537		WP2 uvr A (pKM 101)
+/- S9 mix		-	+	-	+	-	+	-	+	-	+
Negative controls *	Range	10.3 - 26.6	16.9 - 37.9	59.0 - 113.5	69.2 - 124.5	5.6 - 16.2	5.8 - 14.6	4.7 - 10.9	7.2 - 22.1	81.5 - 165.5	16.9 - 199.0
	Mean ± SD	18.5 ± 2.9	27.4 ± 3.6	86.2 ± 10.5	96.8 ± 11.6	10.9 ± 1.8	10.2 ± 1.6	7.8 ± 1.1	14.7 ± 2.4	123.5 ± 16.0	156.3 ± 17.3
Positive controls	Name	2NF	2AA	SAZ	2AA	SAZ	2AA	9AA	2AA	4NQO	2AA
	Range	252.0 - 831.5	217.8 - 750.8	405.6 - 764.3	515.3 - 1818.0	348.3 - 617.7	84.7 - 223.3	144.9 - 767.4	135.0 - 373.1	55.3 - 1152.0	250.1 - 740.9
	Mean ± SD	541.7 ± 111.8	484.3 ± 106.9	584.9 ± 63.0	1166.5 ± 318.4	483.0 ± 48.5	154.0 ± 27.1	456.2 ± 124.1	254.0 ± 49.5	603.6 ± 170.8	495 ± 93.5

* = water for injection, DMSO, acetone, tetrahydrofuran, normal saline injection, sodium phosphate buffer

2AA = 2-aminoanthracene

2NF = 2-nitrofluorene

4NQO = 4-nitroquinoline N-oxide

9AA = 9-aminoacridine

SAZ = sodium azide

Conclusions:

Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jun - 15 Sep 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

no information on pH and osmolality measurements given; only concentrations tested where precipitates were observed

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster lung fibroblasts (CHL/IU cells) were obtained from the Health Science Research Resources Bank, Japan
- Suitability of cells: CHL/IU cells have been recommended in the methods described in OECD guideline 473.
- Doubling time: 15 h
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: Passage 17 was used
- Methods for maintenance in cell culture: The cells were grown in 90-mm diameter petri dishes. Approx. 1.5 x 10E4 cells/mL were seeded and pre-cultured for 2 days prior to each experiment.
- Modal number of chromosomes: 25 per cell
- Periodically checked for karyotype stability: Not specified
- Periodically ‘cleansed’ of spontaneous mutants: Not specified

MEDIA USED
- Type and composition of media: Eagle’s MEM medium (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 0.292 g/L L-glutamine, 1.95 g/L sodium hydrogen carbonate and 10% v/v heat-inactivated newborn calf serum (NBCS, SAFC Biosciences).
- CO2 concentration, humidity level and temperature: The cells were cultured at 37 °C and 5% CO2 in a humidified atmosphere.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Cofactor supplemented post-mitochondrial fraction (S9 mix).
- source of S9 mix: IEDA Trading Corporation (Lot no. CAM202003A)
- method of preparation of S9 mix: S9 mix was prepared from the livers of 7 week old male Sprague Dawley rats. The animals had a body weight in the range of 189 - 239 g and were treated with phenobarbital (30 mg/kg bw for one time, 60 mg/kg bw for three times) and 5,6-benzoflavone (80 mg/kg bw one time).
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentent of the S9 fraction was 24.95 mg/mL. S9 mix contained MgCl2 (5 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in HEPES buffer (4 mM, pH 7.2). The amount of S9 mix in the final culture medium was 6.7%.

Test concentrations with justification for top dose:

Preliminary cell growth inhibition test:
6 h exposure with and without S9 mix: 31.3, 62.5, 125, 500, 1000 and 2000 µg/mL
24 h exposure with and without S9 mix: 31.3, 62.5, 125, 500, 1000 and 2000 µg/mL

Main cytogenicity test:
6 h exposure with and without S9 mix: 500*, 1000* and 2000* µg/mL
24 h exposure without S9 mix: 31.3, 62.5, 125, 500*, 1000* and 2000* µg/mL

*: concentrations selected for chromosome aberration analysis

Justification for top dose: Concentrations of the main cytogenicity test were selected based on the results of the preliminary growth inhibition test.

Vehicle / solvent:

- Vehicle/solvent used: 0.5% w/v methyl cellulose (MC)

- Justification for choice of solvent/vehicle: The test substance was not suspended in distilled water at 20.0 mg/mL and in DMSO and acetone at 200 mg/mL. The test substance was suspended in 0.5 w/v% carboxymethyl cellulose (CMC) solution and 0.5 w/v% MC solution at 20.0 mg/mL. However, the test substance formulation of 20.0 mg/mL prepared with 0.5 w/v% CMC solution was changed to a heterogeneous formulation at room temperature for 2 h after preparation. The test substance formulation of 20.0 mg/mL prepared with 0.5 w/v% MC solution was considered to be stable from the facts that there were no change in color, exothermic reaction nor gas generation at room temperature within 2 h after preparation. Therefore, 0.5 w/v% MC solution was selected as a vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

0.5% MC

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: A single experiment was performed.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 mL cell suspension with 1.5 x 10E4 cells/mL were seeded in 60-mm diameter plastic dishes
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 6 and 24 h
- Harvest time after the end of treatment: 24 h after beginning of treatment

SPINDLE INHIBITOR:
50 µL of a 10 µg/mL demecolcine solution was added to each dish 2 h prior to harvest.

SLIDE PREPRATION AND METAPHASE ANALYSIS:
- Methods of slide preparation and staining technique used including the stain used: The cells were trypsinised, centrifuged, counted, centrifuged again and re-suspended in hypotonic solution (0.075 M potassium chloride) at 37 °C for 15 min. Afterwards, the cells were fixed in methanol : acetic acid (3:1) for three times. The suspension was dropped onto a glass slide, dried and stained with 2 vol% Giemsa solution for about 15 min.
- Number of cells spread and analysed per concentration: 300
- Criteria for scoring chromosome aberrations: Metaphase cells containing 25 ± 2 chromosomes were included in the analysis. The total number of cells with structural aberrations and the number of aberrant cells in each aberration category were recorded. The following categories were investigated: chromatid breaks, chromatid exchanges, chromosome breaks, chromosome exchanges and other structural aberrations.
- Determination of polyploidy: Yes, the number of polyploidy cells with triploid or more (38 chromosomes) was recorded among 300 metaphase cells.
- Determination of endoreplication: Yes, the number of endoreduplicated cells was recorded among 300 metaphase cells.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative doubling population (RDP) and relative increase in cell counts (RICC).

METHODS FOR MEASUREMENTS OF GENOTOXICIY: Frequency of cells with structural chromosome aberrations (excluding gaps) when compared to solvent controls.

Evaluation criteria:

Regarding the frequencies of cells with chromosomal aberrations, the test substance was judged to be negative if:
- All results were inside the distribution of the historical data of the negative control group.
- All results were outside the distribution of the historical data of the negative control group, but none of the doses of the test substance exhibited a statistically significant increase compared with the concurrent negative control.

Regarding the frequencies of cells with chromosomal aberrations, the test substance was judged to be positive if:
- All results were outside the distribution of the historical data of the negative control group.
- There was a statistically significant increase compared with the concurrent negative control.
- The increase of the frequencies of cells with chromosomal aberrations was dose-related.

Statistics:

Statistical methods were performed in order to evaluate the frequencies of cells with
structural aberrations and numerically aberrant cells. Significance level of these tests was both sides of 1% and 5%. Fisher’s exact test was performed in order to compare in the negative control group with positive control group. Fisher’s exact test was performed in order to compare in the negative control group with test substance group when the frequencies of cells with chromosomal aberrations in each test substance group were outside the distribution of the historical data of the negative control group. When at least one of the test substance doses exhibited a statistically significant increase compared with the negative control group in the Fisher’s exact test, the dose-dependency was evaluated by using Cochran-Armitage trend test.

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was noted for all test item concentrations after 6 h exposure with and without S9 mix and after 24 h exposure without S9 mix at the start and the end of treatment and at the end of the incubation period.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation was noted for all test item concentrations after 6 h exposure with and without S9 mix and after 24 h exposure without S9 mix at the start and the end of treatment and at the end of the incubation period.

RANGE-FINDING/SCREENING STUDY:
A preliminary growth inhibition test was performed to identify suitable concentrations for the chromosome aberration test. Single cultures of CHL/IU cells were exposed to test item concentrations in the range of 31.3 – 2000 µg/mL for 6 h with and without S9 mix and for 24 h continuous exposure without S9 mix. The relative doubling population (RDP) and the relative increase in cell count (RICC) were used to assess cytotoxicity.
For all treatment conditions and all concentrations, precipitation of the test substance in the culture medium was noted at the start and the end of treatment and at the time of harvest. There was no cytotoxicity noted for any condition at any concentration, neither with nor without S9 mix. There were abundant metaphases observed at 500, 1000 and 2000 µg/mL. Based on these findings, the concentrations for the chromosome aberration test were selected to be in the range of 500 – 2000 µg/mL.


STUDY RESULTS:
- Results from cytotoxicity measurements: There was no cytotoxicity noted for any test item concentration at any treatment period, neither with nor without S9 mix. For details on relative population doubling (RPD) and relative increase in cell count (RICC), please refer to Table 2 under “Any other information on results incl. tables”.
- Genotoxicity results: When compared to the solvent control, there was no statistically significant increase in the frequency of structural chromosome aberrations for any treatment condition or any concentration. A slight increase in structural chromosome aberrations was noted after 24 h continuous exposure at 1000 µg/mL, which was outside the historical control data of the solvent control. As the increase was not statistically significant when compared to the solvent control, the structural aberration was judged to be negative. The frequencies of numerically aberrant cells at all observation doses of the test substance in all treatment methods were within the range of the historical data of the negative control. For details on results, please refer to Table 2 under “Any information on results incl. tables”.

HISTORICAL CONTROL DATA: For details on the historical control data please refer to Table 1 under “Any other information on materials and methods incl. tables”.

- Positive historical control data: The frequency of cells with structural chromosomal aberrations in the positive control group were within the range of the historical data and showed a statistically significant increase compared with the negative control group.
- Negative (solvent/vehicle) historical control data: The frequency of cells with chromosomal aberrations in the negative control group was within the range of the historical data.

Table 2: Results of the chromosome aberration test

Exposure (h)	S9 mix	Substance	Dose (µg/mL)	Cytotoxicity		Structural chromosome aberrations^#				Numerical chromosome aberrations^#
				RDP	RICC	Excluding gaps		Gaps		Polyploid cells		Total no. of numerical aberrations
						Total no.	Frequency (%)	No. of gaps	Frequency (%)	Total no.	Frequency (%)	Total no.	Frequency (%)
6	-	MC	0.50%	100.0	100.0	3	1	0	0	0	0	0	0
6	-	Test item	500^P	89.8	85.9	5	1.7	0	0	0	0	0	0
6	-		1000^P	92.1	88.4	5	1.7	0	0	1	0.3	1	0.3
6	-		2000^P	63.8	53.8	6	2	0	0	0	0	0	0
6	-	MMC	0.05	78.4	70.4	70**	23.3**	0	0	0	0	0	0
6	+	MC	0.50%	100.0	100.0	2	0.7	0	0	0	0	0	0
6	+	Test item	500^P	85.2	77.7	3	1	0	0	0	0	0	0
6	+		1000^P	82.1	74.0	2	0.7	0	0	1	0.3	1	0.3
6	+		2000^P	81.5	72.6	3	1	0	0	0	0	0	0
6	+	CPA	4	59.9	47.1	73**	24.3**	0	0	0	0	0	0
24	-	MC	0.50%	100.0	100.0	5	1.7	0	0	0	0	0	0
24	-	Test item	500^P	86.7	81.2	9	3	0	0	0	0	0	0
24	-		1000^P	79.4	71.3	10	3.3	2	0.7	0	0	0	0
24	-		2000^P	63.7	52.5	9	3	0	0	0	0	0	0
24	-	MMC	0.05	67.5	56.7	174**	58**	2	0.7	0	0	0	0
#: 300 metaphase cells scored; **: statistically significant, p < 0.01; CPA: cyclophosphamide, positive control +S9 mix; MC: methyl cellulose, solvent; MMC: mitomycin C, positive control -S9 mix; P: precipitation observed at the start and end of treatment and at the time of harvest

Conclusions:

Interpretation of results: negative in CHL/IU cells with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation in bacteria:

Sorbitan tridocosanoate was tested for gene mutation in bacteria (Ames test) according to OECD guideline 471 and in compliance with GLP (Riken Vitamin, 2018). Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA(pKM101) were exposed to the test item, untreated, solvent (THF) and appropriate positive controls in the presence and absence of metabolic activation (S9 mix).

A preliminary test was performed in order to identify suitable concentrations for the main study. As no cytotoxicity was noted up to the highest tested concentration of 5000 µg/plate, the main bacterial mutagenicity study was conducted at concentrations in the range of 313 – 5000 µg/plate. In the main study, two independent pre-incubation tests using triplicate plates per condition were performed. After 48 h of incubation at 37 °C with and without S9 mix, the bacterial background lawn was inspected and the mean number of revertant colonies was counted for each plate.

Precipitation of the test substance in the agar was observed at all concentrations for all strains, with and without S9 mix. There was no evidence for cytotoxicity at any concentration, neither in the presence nor absence of metabolic activation.

In addition, there was no ≥ 2-fold increase in revertant colony numbers of any of the five tester strains observed following treatment with sorbitan tridocosanoate at any dose level, neither in the presence nor absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations.

The number of revertant colonies induced by the solvent control was within the range of the historical control data for each strain, thus demonstrating an acceptable experimental performance. Appropriate positive control compounds showed a strong increase in the number of revertant colonies, confirming the activity of the S9 mix and the validity of the test system.

Thus, based on the results of the present study and under the experimental conditions chosen, the test item is not mutagenic in bacteria with and without metabolic activation.

In vitro cytogenicity in mammalian cells:

In vitro cytogenicity in mammalian cells was investigated in a chromosome aberration test performed in Chinese hamster lung (CHL/IU) cells in accordance with OECD guideline 473 and in compliance with GLP (Riken Vitamin, 2020). Based on the results of a preliminary growth inhibition test, duplicate cultures were exposed to test item concentrations in the range of 500 – 2000 µg/mL for 6 h with and without metabolic activation (S9 mix) and for test item concentrations in the range of 31.3 – 2000 µg/mL for 24 h continuous exposure in the absence of metabolic activation. For all conditions, solvent (0.5% methyl cellulose, MC) and positive controls (0.05 µg/mL mitomycin C (MMC) and 4 µg/mL cyclophosphamide (CPA)) were included. All cell cultures were harvested 24 h after start of exposure. A total of 300 metaphase cells per condition were scored for the presence of structural and numerical chromosome aberrations. In addition, the relative doubling population (RDP) and relative increase in cell count (RICC) of each culture was assessed to determine cytotoxicity.

Precipitation of the test substance in the culture medium was noted for all test item concentrations after 6 h exposure with and without S9 mix and after 24 h exposure without S9 mix at the start and the end of treatment and at the end of the incubation period. There was no cytotoxicity, evident as a reduction of cell growth of > 50% noted for any condition at any concentration, neither in the presence nor absence of S9 mix.

All control substances showed the expected results and were within the range of the laboratory's historical control data. MMC and CPA induced statistically significant increases in the number of aberrant cells, thus proving the functionality of the S9 mix and demonstrating the validity of the test system.

After 24 h of exposure at 1000 µg/mL without S9 mix, the test item showed a slight increase in the number of structural chromosome aberrations, which was outside the historical control range for the negative control. The increase was statistically not significant when compared to solvent control cultures and therefore considered not relevant. Also for the other conditions, the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9 mix. In addition, the frequency of polyploid cells and cells with endoreduplicated chromosomes were not affected, neither with nor without metabolic activation.

Under the conditions of the test, sorbitan tridocosanoate is considered negative for clastogenic activity in CHL/IU cells in vitro with and without S9 mix. 

 

In vitro gene mutation in mammalian cells

The potential of sorbitan tridocosanoate to induce gene mutations in mammalian cells was investigated in an HPRT test, in line with OECD guideline 476 and according the GLP (Riken Vitamin, 2021). Two independent experiments were performed in Chinese Hamster Ovary (CHO) cells. Based on the results of a preliminary cytotoxicity test, duplicate cultures were exposed to test item concentrations of 2.74 – 2000 µg/mL for 5 h in the presence and absence of S9 mix (Experiment 1), for 5 h in the presence of S9 mix (Experiment 2) and for 24 h in the absence of S9 mix (Experiment 2).

Solvent (DMSO) and positive controls (ethylmethane sulphonate (EMS, 0.4 µL/mL for 5 and 24 h of exposure without S9 mix) and 7,12-dimethylbenzanthracene (DMBA, 15 µg/mL for 5 h of exposure with S9 mix) were included in each experiment. After exposure, the cells were incubated for 7 days to allow expression of the mutant phenotype. The expression period was followed by a 7-day selection period, in which the cells were incubated in selection medium containing 20 µg/mL 6-thioguanine (6-TG) prior to determination of the mutant frequency. 

In both experiments, there was no cytotoxicity up to and including the highest concentration of 2000 µg/mL, neither with nor without metabolic activation. However, precipitation of the test substance in the culture medium was noted in both experiments at 24.69 – 2000 µg/mL at the end of treatment in the presence and absence of metabolic activation.

The mutation frequency of the solvent control was within the range of historical control data. The positive control substances EMS and DMBA markedly increased the mutation frequency in CHO cells, thus demonstrating the sensitivity and validity of the test system and confirming the functionality of the S9 mix.

In the first experiment, no statistically significant increase in the mutant frequency was observed at any dose level, neither with nor without S9 mix. In the second experiment after 5 h exposure with S9 mix, a statistically significant increase in mutant frequency was observed at 24.69 µg/mL. The finding was not dose-related, fell within the range of the laboratory’s historical control data and was therefore not considered relevant.

Under the conditions of the test, sorbitan tridocosanoate was considered negative for gene mutation in CHO cells with and without metabolic activation in vitro.  

 

Conclusion:

In conclusion, assessment of the available experimental data on gene mutation in bacteria, gene mutation in mammalian cells and chromosome aberration in mammalian cells suggests that sorbitan tridocosanoate is neither mutagenic nor clastogenic in vitro.

Justification for classification or non-classification

The available data on genetic toxicity in vitro do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.