Sorbitan, tridocosanoate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Sorbitan, tridocosanoate

EC Number:

301-037-8

EC Name:

Sorbitan, tridocosanoate

Cas Number:

93980-59-7

Molecular formula:

C72H138O8

IUPAC Name:

Sorbitan tridocosanoate

Test material form:

solid: particulate/powder

Method

Target gene:

HGPRT

Species / strain

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.

Controls

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.

Results and discussion

Test results

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”.

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

 

Applicant's summary and conclusion

Conclusions:

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