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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a Key and Supporting Ames test performed according to OECD 471 guideline and GLP principles, the registered substance was found to be not mutagenic with or without metabolic activation when tested in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 up to and including cytotoxic concentrations .

In a Key in vitro Micronucleus test performed according to OECD 487 guideline and GLP principles, the registered substance was found to be not clastogenic and aneugenic with or without metabolic activation when tested in peripheral human lymphocytes up to and including cytotoxic concentrations.

In a Key in vitro Mammalian gene mutation test performed according to OECD 476 guideline and GLP principles, the registered substance was found to be not mutagenic with or without metabolic activation when tested in CHO K1 Chinese hamster ovary cells up to and including cytotoxic concentrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The test was conducted according to OECD/EC guidelines and GLP principles. Test substance applied as received.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
including METI, MHLW and MAFF
Deviations:
no
Principles of method if other than guideline:
No info on version of guidelines used (year).
GLP compliance:
yes (incl. QA statement)
Remarks:
issued 2 December 2002
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine or tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (prepared at SPL from livers of rats exposed to phenobarbitone/ beta-naphtoflavone)
Test concentrations with justification for top dose:
Experiment 1:
Salmonella strains: 15, 50, 150, 500, 1500, 5000 µg/ plate;
E.coli strain: 50, 150, 500, 1500, 5000 µg/ plate.
Experiment 2:
TA100, TA1535: 15, 50, 150, 500, 1500, 5000 µg/ plate;
WP2uvrA, TA98, TA1537: 50, 150, 500, 1500, 5000 µg/ plate.
Vehicle / solvent:
- Vehicle used: Water
- Justification for choice of vehicle: The test material was fully miscible in distilled water at 50 mg/ml in solubility checks performed at SPL.
- The test material was accurately weighed and approximate half-log dilutions were prepared in sterile water by mixing on a vortex mixer on the day of each experiment.
- Formulations adjusted for the stated water content (water content = 51% of the test material).
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/ plate (TA100), 5 µg/ plate (TA1535), 2 µg/ plate (WP2uvrA); 9-Aminoacridine: 80 µg/ plate (TA1537); 4-Nitroquinoline-1-oxide: 0.2 µg/ plate (TA98)
Remarks:
without S9-mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
2-Aminoanthracene: 1 µg/ plate (TA100), 2 µg/ plate (TA1535 and TA1537), 10 µg/ plate (WP2uvrA); Benzo(a)pyrene 5 µg/ plate (TA98)
Remarks:
with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 3

Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 5000 µg/ plate, for TA100 and TA1535 (+/- S9-mix).
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST:
The test material was toxic to TA100 at 5000 µg/ plate (sparse bacterial background lawn) with and without S9-mix. The test substance was not toxic to WP2uvrA when tested up to and including 5000 µg/ plate.

No test material precipitate was observed on the plates at any of the doses tested with or without S9 -mix.

Conclusions:
Interpretation of results: negative

In an Ames test performed according to OECD/ EC guidelines and GLP principles, product XSM 2697 (CT-825-05; a 50% solution of disodium 4-isodecyl sulfosuccinate) was found to be not mutagenic with or without metabolic activation when tested up to and including 5000 µg/ plate.
Executive summary:

An Ames test was performed with product XSM 2697 (CT-825-05; a 50% solution of disodium 4-isodecyl sulfosuccinate) according to OECD/ EC guidelines and GLP principles. The concentration of the test material was adjusted for the water (51%) content and tested up to and including 5000 µg/ plate. The test material was toxic to TA100 at 5000 µg/ plate (sparse bacterial background lawn) with and without S9-mix, but did not show cytotoxicity in the other strains. Appropriate positive controls and vehicle controls were included and gave a response within historical range, demonstrating that test system was valid. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Based on these results, it can be concluded that the test substance was not mutagenic with or without metabolic activation when tested in a reverse mutation assay when tested up to and including cytotoxic concentrations.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 Augustus 2020 - 23 February 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Exception: Concentration, stability, and homogeneity of test item formulations were not determined in this study. However, the test item preparation was performed with approved procedures and documented in detail.
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
S20656-168-A
- Purity, including information on contaminants, isomers, etc.:
99.4% (considered to be 100% for dose calculation). No correction was made for the purity/composition of the test item.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature (taken from label)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The test item formed a yellow homogenous suspension in culture medium.
The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: A solubility test was performed based on visual assessment. The test item formed a yellow homogenous suspension in culture medium. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. In the first and second experiment the test item was crushed and ground in a mortar with a pestle to improve the consistency. Test item concentrations were used within 1 hour after preparation. Any residual volumes were discarded.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No correction was made for the purity/composition of the test item.
A solubility test was performed based on visual assessment. The test item formed a yellow homogenous suspension in culture medium. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. In the first and second experiment the test item was crushed and ground in a mortar with a pestle to improve the consistency. Test item concentrations were used within 1 hour after preparation. Any residual volumes were discarded.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution:
In the first and second experiment the test item was crushed and ground in a mortar with a pestle to improve the consistency.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: In the Dose-range Finding test, the test item precipitated in the culture medium at a concentration of 1000 µg/mL.
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Cultured peripheral human lymphocytes. Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years).
- The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2019) are presented below:
Dose-range finding study: age 28, AGT = 14.3 h
First cytogenetic assay: age 23, AGT = 13.3 h
Cytogenetic assay 1A: age 23, AGT = 13.3 h
Second cytogenetic assay: age 34, AGT = 14.7 h

For lymphocytes:
- Sex, age and number of blood donors: Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years).
- Whether whole blood or separated lymphocytes were used: Whole blood (0.4 mL)
- Whether blood from different donors were pooled or not: Not specified
- Mitogen used for lymphocytes: 0.1 mL (9 mg/mL) Phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel Europe Ltd., Dartford, United Kingdom) was added.
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 45 - 93%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 32.8 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium, was used as solvent for the positive controls.
Cytokinesis block (if used):
Cytochalasine B (Sigma; 5 µg/mL)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life Technologies).
The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium : Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Not specified.
Test concentrations with justification for top dose:
The highest concentration analyzed was selected based on the solubility of the test item in the culture medium.
-In the dose-range finding test, blood cultures were treated with 31, 63, 125, 250, 500 and 1000 µg test item/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.
-Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay: Without and with S9-mix: 250, 500 and 1000 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).
-To obtain more information about the possible clastogenicity and aneugenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay: Without S9-mix: 250, 500 and 1000 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).




Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640 medium (culture medium, Life Technologies, Bleiswijk, The Netherlands).
Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium, was used as solvent for the positive controls.
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium
Positive controls:
yes
Remarks:
Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium was used as solvent
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Remarks:
Mitomycin Cwas used as a direct acting clastogen. Colchicine was used as a direct acting aneugen. Cyclophosphamide was used as an indirect acting clastogen, requiring metabolic activation.
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate
- Number of independent experiments 2 (First and Second cytogenetic assay)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium (RPMI 1640 medium)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
First cytogenetic assay (in the absence and presence of S9-fraction): 3 hours exposure
Second cytogenetic assay (in the absence of S9-mix): 24 hours exposure
- Harvest time after the end of treatment (sampling/recovery times):
First cytogenetic assay (in the absence and presence of S9-fraction): 27 hours harvest time
Second cytogenetic assay (in the absence of S9-mix): 24 hours harvest time

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
Cytochalasine B (Sigma; 5 µg/mL) 24 hours exposure
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with Charles River Den Bosch study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In cytogenetic assay 1 and 2, also mononucleated cells were scored for the presence of micronuclei. These results are retained in the raw data but not reported. Since the lowest concentration of MMC-C and CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei. Due to cytotoxicity the number of examined binucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase was observed, this has no effect on the study integrity.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The following criteria for scoring of binucleated cells were used:
•Main nuclei that were separate and of approximately equal size.
•Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
•Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
•Trinucleated, quadranucleated, or multinucleated cells.
•Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
•The diameter of micronuclei should be less than one-third of the main nucleus.
•Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
•Micronuclei should have similar staining as the main nucleus.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cytokinesis-block proliferation index (CBPI index)
- Any supplementary information relevant to cytotoxicity: A minimum of 500 cells (with a maximum deviation of 5%) per culture were counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}

((No. mononucleate cells)+(2×No. binucleate cells)+(3×No. multinucleate cells))
CBPI = -------------------------------------------------------------------------------
(Total number of cells)

t = test item or control treatment culture
c = vehicle control culture
Three analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. The test item was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
During or after exposure of the stimulated human lymphocytes to the test item, cells were cultured to allow chromosome or spindle damage to lead to the formation of micronuclei in interphase cells. Micronuclei are small particles consisting of acentric chromosome fragments (clastogenic event) or whole chromosomes (aneugenic event leading to chromosome loss), which are unable to migrate to the poles during the anaphase stage of cell division. After telophase, these fragments may not be included in the nuclei of daughter cells and form single or multiple micronuclei in the cytoplasm.

Evaluation criteria:
ACCEPTABILITY CRITERIA
An in vitro micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Key result
Species / strain:
lymphocytes: cultured human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
First cytogenetic assay: 3 h exposure, 27 h harvest
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
The test item was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: cultured human peripheral lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
Second cytogenetic assay: 24 h exposure, 24 h harvest
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
The test item was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (if applicable): In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test. The test item was tested in the absence and presence of S9-mix.
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma; 5 µg/mL) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was determined by the solubility of the test item in the culture medium.
After 3 hours exposure to the test item in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
Cytotoxicity of the test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was determined by the solubility.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
Distribution historical positive control data from experiments performed between November 2017 and November 2020.
*Binucleated cells, -S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 46.7
SD: 20.3
n: 96
Lower Control Limit (95% Control Limits): 7
Upper Control Limit (95% Control Limits): 87

*Binucleated cells, -S9 mix, 24 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 41.4
SD: 17.4
n: 98
Lower Control Limit (95% Control Limits): 7
Upper Control Limit (95% Control Limits): 75

*Binucleated cells, +S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 33.0
SD: 13.9
n: 105
Lower Control Limit (95% Control Limits): 6
Upper Control Limit (95% Control Limits): 60

SD = Standard deviation
n = Number of observations
- Negative (solvent/vehicle) historical control data:
Distribution historical negative control data from experiments performed between November 2017 and November 2020.
*Binucleated cells, -S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 5.8
SD: 4.1
n: 94
Lower Control Limit (95% Control Limits): -2
Upper Control Limit (95% Control Limits): 14
*Binucleated cells, -S9 mix, 24 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 5.5
SD: 4.1
n: 91
Lower Control Limit (95% Control Limits): -3
Upper Control Limit (95% Control Limits): 13
*Binucleated cells, +S9 mix, 3 hours exposure:
Mean number of micronucleated cells (per 2000 cells): 6.2
SD: 4.1
n: 97
Lower Control Limit (95% Control Limits): -2
Upper Control Limit (95% Control Limits): 14
SD = Standard deviation
n = Number of observations

Table 1.  Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with Disodium isodecyl sulfosuccinate (CAS No. 37294-49-8) in the Dose-range Finding Test


Without metabolic activation (-S9-mix)

 

 

3 hours exposure time, 27 hours harvest time

 

 

 

Concentration µg/mL

Number of cells with ….nuclei

CBPI

% cytostasis 

 

1

2

3 or more

 

0

148

296

62

1.83

0

 

31

169

273

64

1.79

5

 

63

207

260

46

1.69

17

 

125

202

251

48

1.69

17

 

250

200

267

43

1.69

17

 

500

201

260

42

1.68

18

 

  10001)

210

271

26

1.64

23

 

 

With metabolic activation (+S9-mix)

 

 

3 hours exposure time, 27 hours harvest time

 

 

 

Concentration µg/mL

Number of cells with ….nuclei

CBPI

% cytostasis 

 

1

2

3 or more

 

0

179

271

62

1.77

0

 

31

187

265

51

1.73

5

 

63

180

272

49

1.74

4

 

125

195

263

43

1.70

10

 

250

193

240

69

1.75

2

 

500

168

274

62

1.79

-2

 

  10001)

205

267

51

1.71

9

 

 

Without metabolic activation (-S9-mix)

 

 

24 hours exposure time, 24 hours harvest time

 

 

 

Concentration µg/mL

Number of cells with ….nuclei

CBPI

% cytostasis 

 

1

2

3 or more

 

0

156

299

55

1.80

0

 

31

201

243

59

1.72

11

 

63

222

255

33

1.63

22

 

125

243

208

51

1.62

23

 

250

225

236

41

1.63

21

 

500

214

262

32

1.64

20

 

  10001)

257

228

18

1.52

35

 

 

Note: All calculations were performed without rounding off.

1)  The test item precipitated in the culture medium

Table 2.  Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated with Disodium isodecyl sulfosuccinate (CAS No. 37294-49-8) in the First Cytogenetic Assay and Cytogenetic Assay 1A

Without metabolic activation (-S9-mix)

 

 

 

3 hours exposure time, 27 hours harvest time

 

 

 

 

 

 

 

Concentration µg/mL

CBPI

Mean CBPI

% cytostasis 

0

1.79

-

1.84

1.81

0

250

1.69

-

1.75

1.72

11

500

1.73

-

1.74

1.73

10

1000

1.63

-

1.75

1.69

15

0.25 MMC-C

1.48

-

1.50

1.49

40

0.38 MMC-C

1.39

-

1.42

1.40

50

0.1 Colch

1.14

-

1.16

1.15

82

 

 

 

 

 

 

With metabolic activation (+S9-mix)

 

 

 

3 hours exposure time, 27 hours harvest time

 

 

 

 

 

 

 

Concentration µg/mL

CBPI

Mean CBPI

% cytostasis 

0

1.99

-

2.01

2.00

0

250

1.98

-

2.02

2.00

0

500

1.99

-

2.03

2.01

-1

1000

1.92

-

1.95

1.94

7

15CP

1.45

-

1.47

1.46

54

17.5CP

1.40

-

1.41

1.41

60

 

 

Note: All calculations were performed without rounding off.

Table 3. Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with Disodium isodecyl sulfosuccinate (CAS No. 37294-49-8) in the First Cytogenetic Assay and Cytogenetic Assay 1A

Without metabolic activation (-S9-mix)

3 hours exposure time, 27 hours harvest time

 

Concentration (µg/mL)

Cytostasis (%)

Number of binucleated cells with micronuclei1)

 

1000

1000

2000

 

A

B

A+B

 

0

0

1

1

2

 

250

11

0

1

1

 

500

10

0

2

2

 

1000

15

1

2

3

 

0.25-C

40

8

16

    24****

 

0.1 Colch

82

16

18

    34****

 

 

With metabolic activation (+S9-mix)

3 hours exposure time, 27 hours harvest time

 

Concentration (µg/mL)

Cytostasis (%)

Number of binucleated cells with micronuclei1)

 

1000

1000

2000

 

A

B

A+B

 

0

0

3

5

8

 

250

0

2

3

5

 

500

-1

6

3

9

 

1000

7

4

6

10

 

15 CP

54

28

32

    60****

 

*)  Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.

1)   1000 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

Table 4.  Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with Disodium isodecyl sulfosuccinate (CAS No. 37294-49-8) in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix)

 

 

 

24 hours exposure time, 24 hours harvest time

 

 

 

 

 

 

 

Concentration µg/mL

CBPI

Mean CBPI

% cytostasis 

0

1.58

-

1.60

1.59

0

250

1.50

-

1.51

1.50

14

500

1.41

-

1.52

1.47

21

1000

1.41

-

1.45

1.43

26

0.15 MMC-C

1.26

-

1.27

1.27

54

0.23 MMC-C

1.17

-

1.21

1.19

67

0.05 Colch

1.02

-

1.03

1.02

96

 

 

Note: All calculations were performed without rounding off.

Table 5.  Number Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with Disodium isodecyl sulfosuccinate (CAS No. 37294-49-8) in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix)

24 hours exposure time, 24 hours harvest time

 

Concentration (µg/mL)

Cytostasis (%)

Number of binucleated cells with micronuclei1)

 

1000

1000

2000

 

A

B

A+B

 

0

0

0

1

1

 

250

14

0

1

1

 

500

21

1

0

1

 

1000

26

1

2

3

 

0.15-C

54

5

7

 12**

 

0.05 Colch

96

82)

42)

 12****

 

*)  Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.

1)   1000 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

2)   318 and 321 binucleated cells were scored for the presence of micronuclei, respectively.

Conclusions:
Disodium isodecyl sulfosuccinate (CAS No. 37294-49-8) is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate Disodium isodecyl sulfosuccinate (CAS No.37294-49-8) for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments.


The study procedures described in this report are in compliance with the most recent OECD guideline.


The vehicle of the test item was culture medium.


In the first cytogenetic assay, the test item was tested up to 1000 μg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. The test item precipitated in the culture medium at this dose level.


In the second cytogenetic assay, the test item was again tested up to 1000 μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix.


The number of binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.


The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. The positive control chemical colchicine produced a statistically significant increase in the number of binucleated cells with micronuclei in at least one experiment. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.


The test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.


In conclusion, this test is valid and Disodium isodecyl sulfosuccinate (CAS No. 37294-49-8) is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 September 2020 to ...
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 29 July 2016
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Official Journal L 142, 31/05/2008
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
S20656-168-A
- Purity, including information on contaminants, isomers, etc.:
99.4% (considered to be 100% for dose calculation). No correction was made for the purity/composition of the test item.
-Expiry date: 30 June 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Controlled room temperature (15-25°C, ≤70% relative humidity)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and short period of the study.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Based on the available information about the test item (Safety Data Sheet) the test item is soluble in water (≥ 485 g/l (20 °C) (pH: 5.5)). Therefore, trial formulation was also performed at the Test Facility at the concentration of 200 mg/mL using distilled water and the obtained formulation was an opalescence solution. Therefore, distilled water was selected as vehicle for this study in agreement with the Sponsor. This vehicle (solvent) is compatible with the survival of the cells and the S9 activity.
The test item was formulated in the selected vehicle (solvent) to provide a suitably concentrated stock solution as follows. The necessary amount of test item was weighed into a calibrated volumetric flask (no correction for purity of the test item was applied). Approximately 80% of the required volume of vehicle (solvent) was added and the formulation was stirred until homogeneity was reached (It was ultrasonicated 10 minutes), then the volume was adjusted to the required final level. From the stock solution, several dilutions were prepared using the selected vehicle (solvent) to prepare dosing solutions for lower doses. The vehicle (solvent) was sterile in each case. The stock solutions as well as all dilutions (dosing solutions) were prepared freshly at the beginning of the experiments in the testing laboratory in a sterile hood.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: The pH and osmolality of the treatment medium at the end of the treatment was determined for all concentrations.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: The pH and osmolality of the treatment medium at the end of the treatment was determined at all concentrations. In Assay 1 and 2, there were no large changes in pH and osmolarity after treatment in any cases.
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (Hprt) enzyme locus in CHO K1 Chinese hamster ovary cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO K1
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: The CHO cell line was originally derived from the ovary of a female Chinese hamster (Puck and Kao, 1967). The CHO K1 is a sub-line of CHO cell line. The CHO K1 cell line was purchased from American Type Culture Collection (ATCC).

For cell lines:
- Absence of Mycoplasma contamination: Checking of mycoplasma infection was carried out for each batch of frozen stock; the cell line was tested negative.
- Periodically ‘cleansed’ of spontaneous mutants: yes. Prior to use in this test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium on 22 April 2016


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
For each experiment, one or more vials were thawed rapidly, the cells were diluted in F12-10 medium (“culture medium”) and incubated at 37°C (±0.5°C) in a humidified atmosphere (5±0.3% CO2 in air). When cells were growing well, subcultures were established in an appropriate number of flasks. Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA) solution was used for cell detachment to subculture.
Four types of Ham's F12 medium were prepared as follows
1) Final concentration in F12-1:
Foetal bovine serum (FBS, heat inactivated): 1 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *0.01 mL/mL
2)Final concentration in F12-5:
Foetal bovine serum (FBS, heat inactivated): 5 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *: 0.01 mL/mL
3)Final concentration in F12-10:
Foetal bovine serum (FBS, heat inactivated): 10 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *: 0.01 mL/mL
4)Final concentration in F12-SEL**:
Foetal bovine serum (FBS, heat inactivated): 10 % v/v
L-Glutamine: 0.01 mL/mL
Antibiotic-Antimycotic solution *: 0.01 mL/mL

*: Standard content of the antibiotic-antimycotic solution is 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B.
**: Hypoxanthine-free Ham’s F-12 medium was used for preparation of the selection culture medium
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Male Wistar rats (444-628 g animals were 17-20 weeks old at the initiation, CRL code: E13142) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg bw/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Initiation date of the induction of liver enzymes used for preparation S9 used in this study was 02 September 2019 (CRL code: E13142).
On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized.
Homogenates were centrifuged for 10 minutes at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fraction for this study was 05 September 2019 (Expiry date: 05 September 2021).
The protein concentration of the preparation was determined by a chemical analyser at 540 nm in the Clinical Chemistry Laboratory of the test Facility. The protein concentration of the S9 fraction used in the study was determined to be 24.5 g/L. The sterility of the preparation was confirmed.

- method of preparation of S9 mix: The S9-mix was prepared as follows:
a) Concentration of the stock solution:
HEPES* 20 mM
KCl 330 mM
MgCl2 50 mM
NADP** 40 mM
D-Glucose 6 phosphate (Monosodium salt) 50 mM
F12-10 -
S9 fraction -
b) Concentration in the mix:
HEPES* 0.2 mL/mL
KCl 0.1 mL/mL
MgCl2 0.1 mL/mL
NADP** 0.1 mL/mL
D-Glucose 6 phosphate (Monosodium salt) 0.1 mL/mL
F12-10 0.1 mL/mL
S9 fraction 0.3 mL/mL
*HEPES = N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**NADP= β-Nicotinamide-adenine dinucleotide-phosphate

Prior to addition to the culture medium the S9-mix was kept in an ice bath.
- concentration or volume of S9 mix and S9 in the final culture medium:
For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to 9 mL of cell culture medium to give a total of 10 mL (the same ratio was applied in those cases when higher treatment volume was used). The final concentration of the liver homogenate in the test system was 3%.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens (2-Aminoanthracene and Benzo(a)pyrene), that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study functioned appropriately.
Test concentrations with justification for top dose:
Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:
Assay 1
5-hour treatment in the presence of S9-mix: 1500, 1250, 1000, 750, 500, 250 and 125 µg/mL
5-hour treatment in the absence of S9-mix: 500, 450, 400, 350, 300, 250 and 125 µg/mL.
Assay 2
5-hour treatment in the presence of S9-mix: 1500, 1250, 1000, 750, 500, 250 and 125 µg/mL
24-hour treatment in the absence of S9-mix: 900, 800, 700, 600, 500, 250 and 125 µg/mL.
Assay 3
5-hour treatment in the absence of S9-mix: 600, 575, 550, 525, 500, 450, 400, 250 and 125 µg/mL.
24-hour treatment in the absence of S9-mix: 700, 675, 650, 625, 600, 500, 250, 125 and 62.5 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water (test substance)
Vehicle for the positive controls: DMSO

- Justification for choice of solvent/vehicle: Based on the available information about the test item (Safety Data Sheet) the test item is soluble in water (≥ 485 g/l (20 °C) (pH: 5.5)). Therefore, trial formulation was also performed at the Test Facility at the concentration of 200 mg/mL using distilled water and the obtained formulation was an opalescence solution. Therefore, distilled water was selected as vehicle for this study in agreement with the Sponsor. This vehicle (solvent) is compatible with the survival of the cells and the S9 activity.

- Justification for percentage of solvent in the final culture medium:
Untreated negative controls:
yes
Remarks:
HAM F12-1 (5 h -S9); HAM F12-5 (5 h +S9 and 24 h -S9)
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
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 cultures (in the main tests)
cytotoxicity (plate for survival): triplicate
mutagenicity: for selection of mutants 5 replicate plates; for viability (CE) 3 replicate plates.)
- Number of independent experiments; 3 (Assay1, Assay 2, Assay 3)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): at least 2x10E6 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Assay 1: 5 hours exposure (with and without metabolic activation)
Assay 2: 5 hours exposure with metabolic activation and 24 hours exposure without metabolic activation
Assay 3: 5-hour and 24-hour treatment without metabolic activation; this was decided on the basis of results of the Assay 1 and Assay 2 and according to the OECD No. 476 guideline instructions (up to the cytotoxicity limit).
- Harvest time after the end of treatment (plating for survival):
5 hours exposure experiments: 19 h incubation
24 hours exposure experiment: 0 h incubation

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
Assay 1, 2 and 3: 7 days expression period
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells):
Survival: 6 days (1 d treatment or 5 h treatment and 19 h incubation; 5 d colony growing)
Viability: 13 days (1 d treatment or 5 h treatment and 19 h incubation; 7 d expression; 5 d colony growing)
Mutagenicity: 15 days ((1 d treatment or 5 h treatment and 19 h incubation; 7 d expression; 7 days colony growing in selective medium
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
6-thioguanine, 10 µg/mL, 7 days cell exposure for colony growing in selective medium
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
viability: adjust cell number to 4x10E5 cells/mL, then diluted to 40 cells/mL
mutant phenotype: adjust cell number to 4x10E5 cells/mL

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative survival (RS)
Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.

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.
Rationale for test conditions:
The concentrations selected for the main experiments were based on results of the performed Preliminary Toxicity Test according to the OECD No. 476 guideline instructions (up to the cytotoxicity limit).
Evaluation criteria:
ASSAY ACCEPTANCE CRITERIA
The assay was considered valid if all of the following criteria were met (based on the relevant guidelines):
1. The mutant frequency in the negative (vehicle/solvent) control cultures was in accordance with the general historical control data.
2. The positive control chemicals induced a statistically significant increase in mutant frequency and should be within the historical data for positive controls.
3. The cloning efficiency of the negative controls was in the range of 60-140% on Day 1 and 70-130% on Day 8; under these conditions an adequate number of cells are analysable.
4. At least four test item concentrations in duplicate cultures were presented.

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:
Relative survivals were assessed by comparing the cloning efficiency of the treated groups to the negative (vehicle/solvent) control.
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.
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 non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney 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
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: In Assay 1,2 and 3, there were no large changes in pH after treatment in any cases.
- Data on osmolality: In Assay 1,2 and 3, there were no large changes in osmolality after treatment in any cases.
- Precipitation and time of the determination:
In Assay 1 and 2, insolubility (minimal amount of precipitate) was detected at 1500 – 500 µg/mL concentration range in the final treatment medium at the end of the treatment in the experiments with metabolic activation and no insolubility was detected in the final treatment medium at the end of the treatment in the experiments without metabolic activation. The precipitation did not interfere with the reading of the results. In Assay 3, no insolubility was detected in the final treatment medium at the end of the treatment in the experiments without metabolic activation.

RANGE-FINDING/SCREENING STUDIES (if applicable):
Treatment concentrations for the mutation assay were selected based on the results of a short preliminary experiment. 5-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix were performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest test concentration in the preliminary test was 2000 µg/mL (the recommended maximum concentration).
Insolubility (precipitate or minimal amount of precipitate) was detected in the preliminary experiment in the final treatment medium at the end of the treatment at 2000 and 1000 µg/mL concentration range in 5-hour treatment in the presence of S9-mix and at 1000 µg/mL concentration range in 24-hour treatment in the absence of S9. The concentrations selected for the main experiments were based on results of the performed Preliminary Toxicity Test according to the OECD No. 476 guideline instructions (up to the cytotoxicity limit). Seven concentrations were selected for the main experiments.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays, and the observed values were in the expected range (5-20 x 10E-6) as shown in the OECD No. 476 guideline.
The positive controls (DMBA in the presence of metabolic activation and EMS in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays.
The cloning efficiencies for the negative (vehicle) controls on Days 1 and 8 were within the target range of 60-140% and 70-130% in all assays.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency
In Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest evaluable concentration of 1000 µg/mL concentration showed a relative survival (RS) of 5% and 32% on Day 1 and Day 6 respectively). The degree of cytotoxicity at this concentration was considered to be acceptable.
In Assay 1, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest evaluable concentration of 500 and 450 µg/mL concentrations showed a Day 6 relative survival of 48% and 53%, respectively). Since the Day 1 RS% was 1% and 2% respectively, higher dose concentrations were not achievable.
In Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (the highest concentration of 1500, 1250 and 1000 µg/mL showed a Day 6 relative survival of 1%, 12% and 58%, respectively).
In Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (the highest evaluable concentration of 600 µg/mL concentration showed a Day 6 relative survival of 39%).
In Assay 3, in the absence of S9-mix (5-hour treatment), similarly to the first assay, marked cytotoxicity of the test item was observed (the highest concentration of 600, 575, 550, 525, 500 and 450 µg/mL showed a Day 6 relative survival of 52%, 61%, 57%, 59% 50% and 52%, respectively). Since the Day 1 RS% was 2% to 3%, higher dose concentrations were not achievable, hence the concentrations selected are fully valid.
In Assay 3, in the absence of S9-mix (24-hour treatment), similarly to the second assay, marked cytotoxicity of the test item was observed (the highest concentration of 700, 675, 650 and 625 µg/mL showed a relative survival of 28%, 48%, 43% and 58%, respectively). Since the Day 1 RS% was 5% 11%, 11% and 28%, higher dose concentrations were not achievable, hence the concentrations selected are adequate.

Note: In the absence of S9-mix (5-hour and 24-hour treatment), the highest selected concentrations did not achieve the target Day 6 cytotoxicity range, which were selected on the basis of results of the preliminary experiment (the guideline criteria is that the dose “should aim to achieve between 20 and 10% RS”). The dose response was very steep, with very good growth of the few remaining cells after the initial (Day 1) cytotoxicity event (hence a relatively high Day 6 RS% even when there was excessive initial cytotoxicity). Hence it is difficult to achieve an exact cytotoxicity with a Day 6 RS% of 10-20%. An additional experiment with the short and long treatment without metabolic activation (Assay 3) was performed with more closely spaced concentrations, as per the guideline, in an attempt to give further information and to confirm the results so far, with the intent of meeting the preferred RS% of 10-20% if possible. The aim was to have a cytotoxicity of approximately 10%-20% (Relative Survival, RS) achieved in at least one concentration level in the assay. Although the relative survival value of the tested highest surviving concentrations in Assay 3 are higher than the recommended range, the cells of the closely spaced (technically feasible and meet the guideline criteria) higher concentration(s) samples did not survive the expression period (due to excessive cytotoxicity) as it was observed in the other parts of this study. Thus, these concentrations were selected as highest evaluated concentration which were harmonised with the obtained results so far and confirmed it, Therefore, it was acceptable.

In other words: In case of short treatment without metabolic activation, the preliminary results showed that there was excessive cytotoxicity at 1000 µg/mL concentration and 2% survival level at 500 µg/mL concentration after treatment (day 1). The obtained survival result in the Assay 1 was similarly 1% at 500 µg/mL concentration and the additional Assay 3 with more closely spaced concentrations gave same 2-3% survival result at 600-500 µg/mL concentration range after treatment (day 1).
In case of long treatment without metabolic activation, the preliminary results showed that there was excessive cytotoxicity at 1000 µg/mL concentration and 53% survival level at 500 µg/mL concentration after treatment (day 1). The obtained survival result in the Assay 2 was similarly excessive cytotoxicity at 900-700 µg/mL concentration range 6% at 600 µg/mL concentration and 40% at 500 µg/mL concentration. The additional Assay 3 with more closely spaced concentrations provided similar 5% survival result at 700 µg/mL concentration and 11-34% at 675-600 µg/mL concentration range after treatment (day 1).
Based on this information, the survival values of the tested highest concentrations were very close to excessive cytotoxicity (where plating for mutagenicity testing is impossible due to the low cell number). Therefore, closer evaluable concentrations could not be selected hence the assay is considered acceptable.

- Genotoxicity results:
See under "Any other information on results incl. tables".
In Assay 1, in the presence of S9-mix (5-hour treatment), an evaluation was made using data of five concentrations: statistically significant increase in the mutation frequency (at p<0.01 level) was observed at the concentration of 750 µg/mL, although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (14.3 x 10E-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10E-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). Therefore, it was concluded as biologically not relevant increase. In the absence of S9-mix (5-hour treatment), an evaluation was made using data of all seven concentrations: no statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).
In Assay 2, in the presence of S9-mix (5-hour treatment), an evaluation was made using data of six concentrations: no statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment). In the absence of S9-mix (24-hour treatment), an evaluation was made using data of four concentrations: no statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).
In Assay 3, in the absence of S9-mix (5-hour treatment), an evaluation was made using data of nine concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment). Moreover, it confirmed the result of the Assay 1.
In Assay 3, in the absence of S9-mix (24-hour treatment), an evaluation was made using data of nine concentrations. Statistically significant increase in the mutation frequency (at p<0.05 level) was observed in this experiment at the concentration of 250 µg/mL, although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (9.3 x 10E-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10E- 6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). Therefore, it was concluded as biologically not relevant increase. Overall, this experiment was concluded as negative and confirmed the result of the Assay 2.
Overall, the experiment was concluded as negative.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: See under “Any other information on results incl. tables”
- Negative (solvent/vehicle) historical control data: See under “Any other information on results incl. tables”

Historical Control Data


(updated on 17 October 2017 using data of GLP studies) 











































































































































































 



Mutation frequency
(Number of 6-TG resistant mutants per 106clonable cells)



 



Untreated control



 



5-hour, S9+



5-hour, S9-



24-hour, S9-



mean



18.3



20.7



19.0



standard deviation



15.1



16.4



17.2



minimum



5.1



5.5



3.3



maximum



64.1



55.5



58.0



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




 


DMSO = Dimethyl sulfoxide


DMBA =7,12-Dimethylbenz[a]anthracene


EMS =Ethyl methanesulfonate


S9+ = in the presence of S9-mix


S9- = n the absence of S9-mix


                         


Table 1. Mutagenicity Results of Assay 1

































































































































S9 mixTreatment period (hours)Study phaseTest item or control concentrationTotal number of coloniesMutant
frequency
+5A11500 µg/mL--
1250 µg/mL--
1000 µg/mL277.8
750 µg/mL4614.3**
500 µg/mL185.3
250 µg/mL266.2
125 µg/mL226.2
Negative control195.6
Negative control for DMBA (DMSO)216.4
Untreated control257.2
Positive control (DMBA)1410414.4**
-5A1500 µg/mL174.6
450 µg/mL164.2
400 µg/mL143.7
350 µg/mL185.3
300 µg/mL175.2
250 µg/mL226.3
125 µg/mL175.2

Negative control


257.4

Negative control for EMS (DMSO)


184.9

Untreated control


205.2

Positive control (EMS)


798279.9**

** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control


A1 = Assay 1


+ = in the presence of S9-mix; DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL


- = in the absence of S9-mix; EMS = Ethyl methanesulfonate, 0.4 µL/mL


Negative (vehicle) control = 1% (v/v) Distilled water


DMSO = Dimethyl sulfoxide


Mutant frequencies refer to 106 clonable cells.


 


Table 2. Mutagenicity Results of Assay 2


































































































































S9 mix



Treatment period (hours)



Study phase



Test item or control concentration



Total number of colonies



Mutant
frequency


+5A21500 µg/mL-

-


1250 µg/mL15

3.9


1000 µg/mL236.9
750 µg/mL144.2
500 µg/mL215.6

250 µg/mL


216.3
125 µg/mL216.6
Negative control195.5
Negative control for DMBA (DMSO)216.4
Untreated control205.8
Positive control (DMBA)1718502.3**
-24A2900 µg/mL--
800 µg/mL--
700 µg/mL--
600 µg/mL154.5
500 µg/mL205.6
250 µg/mL216.1
125 µg/mL165.2
Negative control206.1
Negative control for EMS (DMSO)226.7
Untreated control154.2
Positive control (EMS)1112901.1**

** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control


A2 = Assay 2


+ = in the presence of S9-mix; DMBA = 7,12-Dimethylbenz[a]anthracene, 15 µg/mL


- = in the absence of S9-mix; EMS = Ethyl methanesulfonate, 0.4 µL/mL


Negative (vehicle) control = 1% (v/v) Distilled water


DMSO = Dimethyl sulfoxide


Mutant frequencies refer to 106 clonable cells.


 


Table 3. Mutagenicity Results of Assay 3






















































































































































S9 mix



Treatment period (hours)



Study phase



Test item or control concentration



Total number of colonies



Mutant
frequency


-5A3600 µg/mL27

7.6


575 µg/mL20

5.5


550 µg/mL19

4.7


525 µg/mL308.0
500 µg/mL194.9
450 µg/mL174.8
400 µg/mL235.8

250 µg/mL


246.2
125 µg/mL195.4
Negative control236.5
Negative control for EMS (DMSO)266.8
Untreated control357.0
Positive control (EMS)1142430.1**
-24A3700 µg/mL164.4
675 µg/mL205.2
650 µg/mL204.9
625 µg/mL194.8
600 µg/mL205.0
500 µg/mL205.2
250 µg/mL339.3*
125 µg/mL184.5
62.5 µg/mL226.3
Negative control256.6
Negative control for EMS (DMSO)195.0
Untreated control458.2
Positive control (EMS)827503.1**

* = Statistically siginificant increase (at p< 0.05) compared to the relevant vehicle control


** = Statistically significant increase (at p< 0.01) compared to the relevant vehicle control


 


A3 = Assay 3


- = in the absence of S9-mix; EMS = Ethyl methanesulfonate, 0.4 µL/mL


Negative (vehicle) control = 1% (v/v) Distilled water


DMSO = Dimethyl sulfoxide


Mutant frequencies refer to 106 clonable cells.

Conclusions:
In conclusion, no mutagenic effect of Disodium isodecyl sulfosuccinate was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay. The study was considered valid based on the negative and positive control values.
Executive summary:

An in vitro mammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the Hprt locus to evaluate the potential of Disodium isodecyl sulfosuccinate to cause gene mutation. Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The design of this study was based on the Commission Regulation (EC) No. 440/2008 and OECD No. 476 guideline, and the study was performed in compliance with Charles River Laboratories Hungary Kft. standard operating procedures and with the OECD Principles of Good Laboratory Practice.


Distilled water was used as the vehicle (solvent) of the test item in this study. Treatment concentrations for the mutation assays of the main tests were selected based on the results of a preliminary toxicity test as follows:


 


Assay 1


5-hour treatment in the presence of S9-mix:


1500, 1250, 1000, 750, 500, 250 and 125 µg/mL   


5-hour treatment in the absence of S9-mix:


500, 450, 400, 350, 300, 250 and 125 µg/mL.


Assay 2


5-hour treatment in the presence of S9-mix:


1500, 1250, 1000, 750, 500, 250 and 125 µg/mL


24-hour treatment in the absence of S9-mix:


900, 800, 700, 600, 500, 250 and 125 µg/mL.   


Assay 3


5-hour treatment in the absence of S9-mix:


600, 575, 550, 525, 500, 450, 400, 250 and 125 µg/mL.


24-hour treatment in the absence of S9-mix:


700, 675, 650, 625, 600, 500, 250, 125 and 62.5 µg/mL.


 


In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 day expression period following the treatment) and mutagenicity (colony-forming ability at the end of the 7 day expression period following the treatment, in the presence of 6-thioguanine as a selective agent) was determined.


In Assays 1 and 2, insolubility (minimal amount of precipitate) was detected at 1500 – 500 µg/mL concentration range in the final treatment medium at the end of the treatment in the experiments with metabolic activation and no insolubility was detected in the final treatment medium at the end of the treatment in the experiments without metabolic activation. The precipitation did not interfere with the reading of the results. There were no large changes in pH and osmolality after treatment in any cases.


In Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest evaluable concentration of 1000 µg/mL concentration showed a relative survival (RS) of 5% and 32% on Day 1 and Day 6 respectively). The degree of cytotoxicity at this concentration was considered to be acceptable. An evaluation was made using data of five concentrations. Statistically significant increase in the mutation frequency (at p<0.01 level) was observed in this experiment at the concentration of 750 µg/mL, although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (14.3 x 10-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10-6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). Therefore, it was concluded as biologically not relevant increase. Overall, this experiment was concluded as negative.


In Assay 1, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest evaluable concentration of 500 and 450 µg/mL concentrations showed a Day 6 relative survival of 48% and 53%, respectively). Since the Day 1 RS% was 1% and 2% respectively, higher dose concentrations were not achievable. An evaluation was made using data of all seven concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).


In Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (the highest concentration of 1500, 1250 and 1000 µg/mL showed a Day 6 relative survival of 1%, 12% and 58%, respectively). An evaluation was made using data of six concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).


In Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (the highest evaluable concentration of 600 µg/mL concentration showed a Day 6 relative survival of 39%). An evaluation was made using data of four concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment).


 


Note: In the absence of S9-mix (5-hour and 24-hour treatment), the highest selected concentrations did not achieve the target Day 6 cytotoxicity range, which were selected on the basis of results of the preliminary experiment (the guideline criteria is that the dose “should aim to achieve between 20 and 10% RS”).  The dose response was very steep, with very good growth of the few remaining cells after the initial (Day 1) cytotoxicity event (hence a relatively high Day 6 RS% even when there was excessive initial cytotoxicity). Hence it is difficult to achieve an exact cytotoxicity with a Day 6 RS% of 10-20%. An additional experiment with the short and long treatment without metabolic activation (Assay 3) was performed with more closely spaced concentrations, as per the guideline, in an attempt to give further information and to confirm the results so far, with the intent of  meeting the preferred RS% of 10-20% if possible. The aim was to have a cytotoxicity of approximately 10%-20% (Relative Survival, RS) achieved in at least one concentration level in the assay.


 


In Assay 3, no insolubility was detected in the final treatment medium at the end of the treatment in the experiments without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.


 


In the absence of S9-mix (5-hour treatment), similarly to the first assay, marked cytotoxicity of the test item was observed (the highest concentration of 600, 575, 550, 525, 500 and 450 µg/mL showed a Day 6 relative survival of 52%, 61%, 57%, 59% 50% and 52%, respectively). Since the Day 1 RS% was 2% to 3%, higher dose concentrations were not achievable, hence the concentrations selected are fully valid.


An evaluation was made using data of nine concentrations. No statistically significant increases in the mutation frequency were observed at any examined concentrations when compared to the negative (vehicle) control data and there was no dose response to the treatment (a trend analysis showed no effect of treatment). Moreover, it confirmed the result of the Assay 1.


 


In the absence of S9-mix (24-hour treatment), similarly to the second assay, marked cytotoxicity of the test item was observed (the highest concentration of 700, 675, 650 and 625 µg/mL showed a relative survival of 28%, 48%, 43% and 58%, respectively). Since the Day 1 RS% was 5% 11%, 11% and 28%, higher dose concentrations were not achievable, hence the concentrations selected are adequate. An evaluation was made using data of nine concentrations. Statistically significant increase in the mutation frequency (at p<0.05 level) was observed in this experiment at the concentration of 250 µg/mL, although the observed value was within the general historical control range. Furthermore, the observed mutant frequency (9.3 x 10-6) was within the expected range of the negative control samples according to the relevant OECD guideline (expected range: 5-20 x 10 6). No dose response to the treatment was observed (a trend analysis showed no effect of treatment). Therefore, it was concluded as biologically not relevant increase. Overall, this experiment was concluded as negative and confirmed the result of the Assay 2.


 


Note: Although the relative survival value of the tested highest surviving concentrations in Assay 3 are higher than the recommended range, the cells of the closely spaced (technically feasible and meet the guideline criteria) higher concentration(s) samples did not survive the expression period (due to excessive cytotoxicity) as it was observed in the other parts of this study. Thus, these concentrations were selected as highest evaluated concentration which were harmonised with the obtained results so far and confirmed it, Therefore, it was acceptable.


 


In other words:


In case of short treatment without metabolic activation, the preliminary results showed that there was excessive cytotoxicity at 1000 µg/mL concentration and 2% survival level at 500 µg/mL concentration after treatment (day 1). The obtained survival result in the Assay 1 was similarly 1% at 500 µg/mL concentration and the additional Assay 3 with more closely spaced concentrations gave same 2-3% survival result at 600-500 µg/mL concentration range after treatment (day 1).


 


In case of long treatment without metabolic activation, the preliminary results showed that there was excessive cytotoxicity at 1000 µg/mL concentration and 53% survival level at 500 µg/mL concentration after treatment (day 1). The obtained survival result in the Assay 2 was similarly excessive cytotoxicity at 900-700 µg/mL concentration range 6% at 600 µg/mL concentration and 40% at 500 µg/mL concentration. The additional Assay 3 with more closely spaced concentrations provided similar 5% survival result at 700 µg/mL concentration and 11-34% at 675-600 µg/mL concentration range after treatment (day 1).


 


Based on this information, the survival values of the tested highest concentrations were very close to excessive cytotoxicity (where plating for mutagenicity testing is impossible due to the low cell number). Therefore, closer evaluable concentrations could not be selected hence the assay is considered acceptable


 


The spontaneous mutation frequency of the negative (vehicle) control was in accordance with the general historical control range in all assays. The positive controls gave the anticipated increases in mutation frequency over the controls and were in good harmony with the historical data in all assays. At least four evaluated concentrations were presented in all assays. The cloning efficiencies for the negative controls at the beginning and end of the expression period were within the target range. The evaluated concentration ranges were considered to be adequate (concentrations were tested up to the maximum recommended concentrations or cytotoxic range in each test). The overall study was considered to be valid.


In conclusion, no mutagenic effect of Disodium isodecyl sulfosuccinate was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay. The study was considered valid based on the negative and positive control values.

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

Genetic toxicity in vivo

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

Additional information

A key Ames test was performed with registered substance according to OECD 471 guideline and GLP principles (Bowles, 2005). The concentration of the test material was adjusted for the water (51%) content and tested up to and including 5000 µg/ plate. The test material was toxic to TA100 at 5000 µg/ plate (sparse bacterial background lawn) with and without S9-mix, but did not show cytotoxicity in the other strains. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

A supporting Ames study was performed with same formulation according to OECD guideline 471, but no E.coli-strain was included (Mecchi, 2005). All bacterial strains showed negative responses up to and including 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity and/or precipitation of the test substance was observed. Based on information in other reports on the test substance, the concentration of 50% allow to conclude that disodium isodecyl sulfosuccinate is not mutagenic in the Ames test with or without metabolic activation when tested up to and including 2500 µg/plate.

A key in vitro Micronucleus assay was available with registered substance according to OECD 487 guideline and GLP principles using human peripheral lymphocytes both in the presence and absence of metabolic activation, employing 2 exposure times without S9 mix: 3 and 24 hours, and 1 exposure time with S9 mix: 4 hours (de Jong, 2021). In the first cytogenetic assay, the test item was tested up to 1000 μg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. The test item precipitated in the culture medium at this dose level. In the second cytogenetic assay, the test item was again tested up to 1000 μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix.The number of binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The test conditions were adequate and the test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. In conclusion, the registered substance is not clastogenic or aneugenic in human lymphocytes under the experimental conditions of the study.

A key in vitro gene mutation assay was conducted with registered substance according to OECD 476 guideline and GLP principles using cultured CHO K1 Chinese hamster ovary cells at the Hprt locus both in the presence and absence of metabolic activation (Kovács, 2021). Treatments were carried out for 5 hours with and without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). In Assay 1, 5-hour treatment in the presence of S9-mix was done at 1500, 1250, 1000, 750, 500, 250 and 125 µg/mL and in the absence of S9-mix at 500, 450, 400, 350, 300, 250 and 125 µg/mL. In Assay 2, 5-hour treatment in the presence of S9-mix was done at 1500, 1250, 1000, 750, 500, 250 and 125 µg/mL and 24-hour treatment in the absence of S9-mix was done at 900, 800, 700, 600, 500, 250 and 125 µg/mL. In Assay 3, 5-hour treatment in the absence of S9-mix was done at 600, 575, 550, 525, 500, 450, 400, 250 and 125 µg/mL and 24-hour treatment in the absence of S9-mix was done at 700, 675, 650, 625, 600, 500, 250, 125 and 62.5 µg/mL. Precipitation was observed in Assay 1 and 2 but did not interfere with the reading of the results. In Assay 3, no insolubility was detected in the final treatment medium at the end of the treatment in the experiments without metabolic activation. There were no large changes in pH and osmolality after treatment in any cases.
In Assay 1, in the presence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest evaluable concentration of 1000  µg/mL concentration showed a relative survival (RS) of 5% and 32% on Day 1 and Day 6 respectively). The degree of cytotoxicity at this concentration was considered to be acceptable.
 In Assay 1, in the absence of S9-mix (5-hour treatment), marked cytotoxicity of the test item was observed (the highest evaluable concentration of 500 and 450 µg/mL concentrations showed a Day 6 relative survival of 48% and 53%, respectively). Since the Day 1 RS% was 1% and 2% respectively, higher dose concentrations were not achievable.  Although a sporadic statistical significant increase was observed, there were no dose related increases in the mutation frequency, and overall, this experiment was concluded as negative.
In Assay 2, in the presence of S9-mix (5-hour treatment), similarly to the first test, marked cytotoxicity of the test item was observed (the highest concentration of 1500, 1250 and 1000 µg/mL showed a Day 6 relative survival of 1%, 12% and 58%, respectively). In Assay 2, in the absence of S9-mix (24-hour treatment), marked cytotoxicity of the test item was observed (the highest evaluable concentration of 600 µg/mL concentration  showed a Day
 6 relative survival of 39%). There were no dose related increases in the mutation frequency, and overall, this experiment was concluded as negative.
Note: In the absence of S9-mix (5-hour and 24-hour treatment), the highest selected concentrations did not achieve the target Day 6 cytotoxicity range between 20 and 10% Relative survival.
 The dose response was very steep, with very good growth of the few remaining cells after the initial (Day 1) cytotoxicity event (hence a relatively high Day 6 RS% even when there was excessive initial cytotoxicity). Hence it was difficult to achieve an exact cytotoxicity with a Day 6 RS% of 10-20%. An additional experiment with the short and long treatment without metabolic activation (Assay 3) was performed with more closely spaced concentrations, as per the guideline, in an attempt to give further information and to confirm the results so far, with the intent of meeting the preferred RS% of 10-20% if possible. The aim was to have a cytotoxicity of approximately 10%-20% (Relative Survival, RS) achieved in at least one concentration level in the assay.
In Assay 3, in the absence of S9-mix (5-hour treatment), similarly to the first assay, marked cytotoxicity of the test item was observed (the highest concentration of 600, 575, 550, 525, 500 and 450 µg/mL showed a Day 6 relative survival of 52%, 61%, 57%, 59% 50% and 52%, respectively). Since the Day 1 RS% was 2% to 3%, higher dose concentrations were not achievable, hence the concentrations selected are fully valid. In the absence of S9-mix (24-hour treatment), similarly to the second assay, marked cytotoxicity of the test item was observed (the highest concentration of 700, 675, 650 and 625 µg/mL showed a relative survival of 28%, 48%, 43% and 58%, respectively). Since the Day 1 RS% was 5% 11%, 11% and 28%, higher dose concentrations were not achievable, hence the concentrations selected are adequate. Although a sporadic statistical significant increase was observed (5-hour treatment), there were no dose related increases in the mutation frequency, and overall, this experiment was concluded as negative and confirmed the result of the Assay 1 and 2.

Note: Although the relative survival value of the tested highest surviving concentrations in Assay 3 are higher than the recommended range, the cells of the closely spaced (technically feasible and meet the guideline criteria) higher concentration(s) samples did not survive the expression period (due to excessive cytotoxicity) as it was observed in the other parts of this study. Thus, these concentrations were selected as highest evaluated concentration which were harmonised with the obtained results so far and confirmed it, Therefore, it was acceptable.
In conclusion, the test conditions were adequate and no mutagenic effect of registered substance was observed either in the presence or absence of a metabolic activation system under the conditions of this HPRT assay.

Justification for classification or non-classification

The available data do not indicate that disodium isodecyl sulfosuccinate is genotoxic. The substance is thus not classified for genotoxic properties according to CLP Regulation (EC) No. 1272/2008.