Butanedioic acid, sulfo-, 4-C12-14 (even numbered)-alkyl esters, disodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registered test item had no mutagenic activity on the growth of the bacterial strains in a key reverse bacterial mutation study when tested up to 5000 µg/plate or up to cytoxicity with and without metabolic activation.

Read across data from CAS No. 90268-36-3 (Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts) were also available for bacterial and mammalian mutagenicity and chromosomal aberration as supporting studies. In a support Ames test no increase in mutations were observed in different Salmonella typhimurium strains with and without metabolic activation up to cytotoxic concentrations of 316 µg/plate. In a key mammalian gene mutation test in HPRT cells, the test item did not induce mutations up to cytotoxic concentrations of 39.06 or 156.3 µg test item/mL in the absence and presence of metabolic activation, respectively. In a key in vitro Micronucleus study in human peripheral lymphocytes, the test item did not induce chromosomal damage up to a cytotoxic concentration of 156.3 µg/mL in the absence and in the presence of metabolic activation employing two exposure times.

Read-across data from CAS 37294-49-8 (Butanedioic acid, sulfo- [C9-11 (branched)akyl] ester, disodium salts) were available for bacterial and mammalian mutagenicity and a micronucleus assay. In a supporting Ames test performed according to OECD 471 guideline and GLP principles, the read-across 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 supportnig in vitro Micronucleus test performed according to OECD 487 guideline and GLP principles, the read-across 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 supporting in vitro Mammalian gene mutation test performed according to OECD 476 guideline and GLP principles, the read-across 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

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached read-across justification

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No changes in the pH values in the medium were noted.
- Effects of osmolality: No relevant changes in osmolality of the formulations were noted.

RANGE-FINDING/SCREENING STUDIES:
- In the preliminary study cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL in the experiment without and with metabolic activation, respectively. Hence, 39.06 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 156.3 µg/mL in the presence of metabolic activation.
- The next higher concentrations resulted in complete cytotoxicity in the preliminary experiment. In addition, the number of mutants was also already considerable decreased in the main experiments at the highest employed concentrations pointing to general pronounced cytotoxicity. Finding a higher concentration with viable and evaluable cells/mutants was therefore not considered realistic.
- In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
The historical background mutation frequency in this system has been reported to be 1 to 44 mutants per 10 6 survivors in non-activation solvent controls and 6 to 46 per 10 6 survivors in S9 activation solvent controls.
The mutation frequency of the cultures treated with concentrations of 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL culture medium without metabolic activation ranged from 3.78 to 15.74 x 10 -6 clonable cells. These results are within the normal range of the vehicle controls.
The mutation frequency of the cultures treated with concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL culture medium with metabolic activation ranged from 4.44 to 13.04 x 10 -6 clonable cells. These results are within the normal range of the vehicle controls.
The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 475.00 to 780.00 x 10-6 clonable cells in the case of EMS and ranging from 530.71 to 855.00 x 10-6 clonable cells in the case of DMBA, indicating the validity of this test system.
The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6 clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6 clonable cells for EMS and 130.0 to 2693.3 x 106 clonable cells for DMBA.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, the read-across test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

The read-across test item was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix. The read-across test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item.  The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary study test item concentrations of 19.53, 39.06, 78.13, 156.3, 312.5, 625 and 1250 µg/mL medium were employed . Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, 39.06 µg test item/mL were employed as the top concentration for the mutagenicity tests in the absence and 156.3 µg/mL in the presence of metabolic activation.

Main study

Five concentrations 2.44, 4.88, 9.77, 19.53 or 39.06 and 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL were selected for the experiments without and with metabolic activation, respectively.

Cytotoxicity

In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2)was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively.

Experiments without metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 14.35 and 16.67 x 10^-6clonable cells. Hence, the vehicle controls were well within the expected range.

The mutation frequency of the cultures treated with concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 µg test item/mL culture medium ranged from 4.44 to 13.04 x 10^‑6clonable cells. These results are within the normal range of the vehicle controls.

Experiments with metabolic activation

The mutation frequency of the vehicle control aqua ad iniectabilia was 17.87 and 16.05 x 10^-6 clonable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL culture medium ranged from 3.78 to 15.74 x 10^‑6 clonable cells. These results are within the normal range of the vehicle controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 475.00 to 780.00 x 10^-6 clonable cells in the case of EMS and ranging from 530.71 to 855.00 x 10^-6 clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10^-6 clonable cells for the vehicle controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10^-6 clonable cells for EMS and 130.0 to 2693.3 x 10⁶ clonable cells for DMBA.

Under the present test conditions, the read-across test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 2020 to 18 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA 712-C-98-247, August 1998

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted: 21st July 1997, Corrected 26th June, 2020

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli strain)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Male Wistar rats (502-672 g, animals were 5 months old (Charles River Laboratories Hungary code: E13358) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study was 01 September 2020 (Charles River Laboratories Hungary code: E13358).
On Day 4, the rats were euthanized, 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 min 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 dates of preparation of S9 fractions for this study was 04 September 2020 (Charles River Laboratories Hungary code: E13358, Expiry date: 04 September 2022).
- method of preparation of S9 mix :
The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate buffer (**), pH 7.4: 500 mL
Rat liver homogenate (S9): 100 mL
Salt solution for S9 Mix (*): 400 mL

(*) Salt solution for S9 Mix:
NADP Na: 7.66 g
D-glucose-6 phosphate Na: 3.53 g
MgCl2 x 6 H2O: 4.07 g
KCl: 6.15 g
Distilled water: q.s. ad 1000 mL
Sterilization was performed by filtration through a 0.22 μm membrane filter.

(**)0.2 M Sodium Phosphate Buffer, pH 7.4:
Solution A:
Na2HPO4 x 12 H2O: 71.63 g
Distilled water: q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
Solution B:
NaH2PO4 x H2O: 27.6 g
Distilled water: q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
Sodium phosphate buffer pH 7.4:
Solution A: 880 mL
Solution B: 120 mL

- concentration or volume of S9 mix and S9 in the final culture medium :
S9 mix: 500 µL
S9 (rat liver homogenate): 100 mL S9 in 1000 mL S9 mix = 10% v/v = 50 µL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):
The sterility of the preparation was confirmed in each case. The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Charles River Laboratories Hungary Kft. The mean protein concentration of the S9 fraction used was determined to be 21.15 g/L (Charles River Laboratories Hungary code: E13358).
The biological activity in the Salmonella assay of S9 was characterized in each case using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batches of S9 used in this study functioned appropriately.

Test concentrations with justification for top dose:

Preliminary Concentration Range Finding Test: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate with and without S9
Based on the results of the preliminary test, after ultrasonic water bath 100 mg/mL stock solution was prepared in distilled water in Assay 1-2 and 10 mg/mL stock solution was prepared in Assay 3, which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in Assay 1 and Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate, in Assay 3 were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
aqueous solvents (distilled water) for test item and positive controls Sodium azide and Methyl-methanesulfonate
DMSO for the other positive controls

- Justification for choice of solvent/vehicle: In the study two vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive control chemicals.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Distilled Water and DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD) 4 µg/plate in DMSO; TA98 without S9

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 2 µg/plate in DMSO; all Salmonella strains with S9

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 50 µg/plate in DMSO; E. coli P2 uvra with S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (triplicate)
- Number of independent experiments: 3 main tests ( Assay 1, 2 and 3)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation): Preliminary Concentration Range Finding Test and Assay 1
Preincubation: Assay 2 and Assay 3

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes at 37°C in a shaking incubator
- Exposure duration/duration of treatment: 48(±1) hours at 37°C
- Harvest time after the end of treatment (sampling/recovery times): Not applicable

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; lower number of revertant colonies (Significantly lower numbers of revertant colonies are considered to indicate an inhibitory effect where no absent/reduced/slightly/minimal reduced background lawn was observed)
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY
revertant colony numbers per plate, mutation factors (mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate)

Rationale for test conditions:

The experimental methods were conducted according to the methods described by Ames et al. [1] and Maron and Ames [2], Kier et al. [3], Venitt and Parry [4], OECD Guideline No. 471, 1997 [5], Commission Regulation (EC) No. 440/2008, 2008 [6], EPA Guidelines, OPPTS 870.5100, 1998, 1996 [7][8] and according to the relevant SOPs of Charles River Laboratories Hungary Kft.

1. BRUCE N. AMES, JOYCE MCCANN and EDITH YAMASAKI: Methods for Detecting Carcinogens and Mutagens with the Salmonella /Mammalian-Microsome Mutagenicity Test. Mutation Research,31: 347-364, 1975
2. DOROTHY M. MARON and BRUCE N. AMES: Revised Method for the Salmonella Mutagenicity Test. Mutation Research, 113: 173-215, 1983
3. L.D. KIER., Brusick D.J., Auletta, A.E., Von Halle, E.S., Brown, M.M., Simmon, V.F., Dunkel, V., McCann, J., Mortelmans, K., Prival, M., Rao, T.K. and Ray, V: The Salmonella Typhimurium / Mammalian Microsomal Assay. A Report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Research, 168: 69-240, 1986
4. S.VENITT and J.M.PARRY: Mutagenicity Testing, a Practical Approach IRL Press Limited, Eynsham, Oxford England, 1984
5. OECD Guidelines for testing of Chemicals, Section 4, No. 471 “Bacterial Reverse Mutation Test”, Adopted: 21st July 1997, Corrected 26th June, 2020
6Commission Regulation (EC) No. 440/2008, B.13/14. “Mutagenicity: Reverse Mutation Test Using Bacteria”, 30 May 2008
7. EPA Health Effects Test Guidelines, OPPTS 870.5100 “Bacterial Reverse Mutation Test”, EPA 712-C-98-247, August 1998
8. EPA Health Effects Test Guidelines, OPPTS 870.5100 “Escherichia coli WP2 and WP2 uvrA Reverse Mutation Assays”, EPA 712-C-96-247, June 1996 (Public Draft)

Evaluation criteria:

The study was considered valid if:
• the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
• at least five analysable concentrations are presented in all strains of the main tests.
A test item is considered mutagenic if:
• a concentration-related increase in the number of revertants occurs and/or;
• a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
• the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
• the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
A test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft Excel software.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 and 1581 μg/plate in Assay 1

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 and 1581 μg/plate in Assay 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 and 158.1 μg/plate in Assay 3

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 and 1581 μg/plate in Assay 1

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

activation at 5000 and 1581 μg/plate in Assay 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 158.1 and 50 μg/plate in Assay 3

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000**, 1581 and 500 μg/plate in Assay 1; **Note: The background lawn was very faint, therefore it could not be evaluated at 5000 μg/plate .

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000, 1581 and 500 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 158.1 μg/plate in Assay 3

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 and 1581 μg/plate in Assay 1

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 and 1581 μg/plate in Assay 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 158.1 and 50 μg/plate in Assay 3

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without metabolic activation at 5000 μg/plate in Assay 1

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without metabolic activation at 5000 μg/plate in Assay 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The Solubility of the Test Item in distilled water:
• Concentration of test item distilled water 100 mg/mg: clear solution; Solubility in the top solution (test item formulation 50 μL + phosphate buffer 500 μL + top agar 2 mL): slight opalescence; Test item concentration in the test plate 5000 µg/plate
• Concentration of test item distilled water 50 mg/mg: clear solution; Solubility in the top solution (test item formulation 50 μL + phosphate buffer 500 μL + top agar 2 mL): clear solution; Test item concentration in the test plate 2500 µg/plate
- Precipitation and time of the determination:
No precipitate was detected on the plates in the preliminary experiment in both examined bacterial strains with and without metabolic activation.
No precipitate as seen in any examined bacterial strains with and without metabolic activation in the main assays.

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the Preliminary Concentration Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.
Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
No precipitate was detected on the plates in the preliminary experiment in both examined bacterial strains with and without metabolic activation.
Inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development / lower number of revertant colonies*) was observed in Salmonella typhimurium TA98 with and without metabolic activation at 5000 and 2500 μg/plate and in Salmonella typhimurium TA100 with and without metabolic activation at 5000, 2500 and 1000 μg/plate in the preliminary experiment.
*Note: Significantly lower numbers of revertant colonies are considered to indicate

For all test methods and criteria for data analysis and interpretation:
The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Criteria for Validity
The study was considered valid if:
• the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
• at least five analysable concentrations are presented in all strains of the main tests.

Criteria for a Positive Response
A test item is considered mutagenic if:
• a concentration-related increase in the number of revertants occurs and/or;
• a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
• the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
• the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Criteria for a Negative Response
A test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Ames test:
- Signs of toxicity:
• Preliminary Concentration Range Finding Test:
Inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development / lower number of revertant colonies*) was observed in Salmonella typhimurium TA98 with and without metabolic activation at 5000 and 2500 μg/plate and in Salmonella typhimurium TA100 with and without metabolic activation at 5000, 2500 and 1000 μg/plate in the preliminary experiment.
*Note: Significantly lower numbers of revertant colonies are considered to indicate an inhibitory effect where no absent/reduced/slightly/minimal reduced background lawn was observed.
• Mutagenicity Tests (Assay 1)
Inhibitory, cytotoxic effect of the test item (absent /reduced / slightly / minimal reduced background lawn development or lower numbers of revertant colonies*) was observed at the following concentrations:
*Note: Significantly lower numbers of revertant colonies are considered to indicate an inhibitory effect where no absent/reduced/slightly/minimal reduced background lawn was observed.
-in Salmonella typhimurium TA98, TA100 and TA1537 with and without metabolic activation at 5000 and 1581 μg/plate,
-in Salmonella typhimurium TA1535 with and without metabolic activation at 5000**, 1581 and 500 μg/plate,
-in Escherichia coli WP2 uvrA without metabolic activation at 5000 μg/plate
**Note: The background lawn was very faint, therefore it could not be evaluated at 5000 μg/plate .
• Mutagenicity Tests (Assay 2)
Inhibitory, cytotoxic effect of the test item (absent /reduced / slightly / minimal reduced background lawn development or lower numbers of revertant colonies*) was observed at the following concentrations:
*Note: Significantly lower numbers of revertant colonies are considered to indicate an inhibitory effect where no absent/reduced/slightly/minimal reduced background lawn was observed.
-in Salmonella typhimurium TA98, TA100 and TA1537 with metabolic activation at 5000 and 1581 μg/plate,
-in Salmonella typhimurium TA1535 with metabolic activation at 5000, 1581 and 500 μg/plate,
-in Escherichia coli WP2 uvrA without metabolic activation at 5000 μg/plate
• Mutagenicity Tests (Assay 3)
Inhibitory, cytotoxic effect of the test item (absent /reduced / slightly / minimal reduced background lawn development or lower numbers of revertant colonies*) was observed at the following concentrations:
*Note: Significantly lower numbers of revertant colonies are considered to indicate an inhibitory effect where no absent/reduced/slightly/minimal reduced background lawn was observed.
-in Salmonella typhimurium TA98 bacterial strain without metabolic activation at 500 and 158.1 μg/plate;
-in Salmonella typhimurium TA100 bacterial strain without metabolic activation at 158.1 and 50 μg/plate;
-in Salmonella typhimurium TA1535 bacterial strain without metabolic activation at 158.1 μg/plate;
-in Salmonella typhimurium TA1537 bacterial strains without metabolic activation at 158.1 and 50 μg/plate.

Results:
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA100 bacterial strain plate without metabolic activation at 50 μg/plate concentration and at Escherichia coli WP2 uvrA bacterial strain without metabolic activation at 158.1 μg/plate (the observed mutation factor value was: MF: 1.08).
In Assay 2 and Assay 3 (pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA100 bacterial strain at 0.1581 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.22).
In the main assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

Table 1. Historical Control Data (Period of 2011-2019)

Untreated control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	22.0	100.8	12.1	7.9	36.9	28.0	108.3	11.7	9.4	41.9
St. dev.	5.3	18.9	4.4	3.4	10.7	6.7	17.9	3.5	3.7	10.3
Range	9-50	54-210	1-46	1-26	11-82	10-56	65-204	1-39	1-29	16-89
n	2019	2014	2025	2034	2025	2037	2031	2036	2043	2022
DMSO control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	21.2	97.0	12.2	7.8	36.0	27.1	105.8	11.5	9.1	41.0
St. dev.	5.2	18.2	4.4	3.3	10.7	6.7	18.8	3.4	3.6	10.2
Range	6-55	40-217	1-43	1-27	7-81	11-67	53-229	2-33	1-29	9-85
n	2172	2163	2175	2190	2175	2189	2181	2192	2196	2175
Distilled water control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	22.7	100.4	12.2	8.6	38.0	28.6	108.2	11.5	9.9	42.8
St. dev.	5.5	19.7	4.3	3.5	10.6	6.9	19.9	3.3	3.7	10.0
Range	11-45	45-215	2-47	2-24	12-84	10-53	59-222	3-39	1-24	13-91
n	423	2031	2037	429	2055	426	2022	2040	426	2040
DMF control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	20.4	90.2	11.4	7.7	37.3	27.1	98.9	11.1	8.9	40.0
St. dev.	5.2	16.8	4.3	3.4	12.8	6.8	18.0	3.3	3.5	11.0
Range	8-38	54-152	1-34	1-19	16-99	11-49	60-156	3-21	1-23	17-76
n	276	276	276	276	267	276	276	276	273	267
Acetone control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	21.8	97.0	12.2	7.6	37.6	27.6	105.7	11.3	8.7	42.7
St. dev.	4.9	13.9	5.1	2.8	9.6	6.6	13.6	3.2	3.1	9.1
Range	11-39	62-160	4-49	1-17	17-63	15-52	66-177	4-22	1-19	17-70
n	371	375	375	378	369	372	375	378	378	372
Positive reference control data
	without metabolic activation (-S9 Mix)					with metabolic activation (+S9 Mix)
	TA98	TA100	TA1535	TA1537	E. coli	TA98	TA100	TA1535	TA1537	E. coli
Mean	372.6	1203.6	1167.1	442.2	1038.2	2411.0	2424.7	228.3	218.9	255.4
St. dev.	97.9	179.2	172.9	141.2	136.1	267.8	246.9	112.5	47.3	94.7
Range	152-2336	536-2120	208-2440	149-2104	488-2496	312-4918	1116-5240	101-2216	117-838	125-2512
n	2019	2013	2025	2034	2028	2037	2034	2040	2043	2022

TA98:Salmonella typhimuriumTA98, TA100:Salmonella typhimuriumTA100, TA1535:Salmonella typhimuriumTA1535, TA1537:Salmonella typhimuriumTA1537, E. coli:Escherichia coliWP2uvrA;n: number of cases

Table 2: Summary Table of the Assay 1

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	17.3	18.7	89.3	88.3	11.7	12.0	11.3	13.3	46.0	51.0
	MF	1.08	0.98	1.10	0.96	0.97	0.92	0.89	0.98	1.01	1.02
DMSO control	Mean	17.0	19.0	--	91.7	--	12.0	11.0	13.3	--	51.3
	MF	1.06	1.00	--	1.00	--	0.92	0.87	0.98	--	1.03
Distilled water control	Mean	16.0	19.0	81.3	91.7	12.0	13.0	12.7	13.7	45.3	50.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	0.0	0.0	0.0	4.3	0.0	0.0	0.0	0.3	22.7	45.0
	MF	0.00	0.00	0.00	0.05	0.00	0.00	0.00	0.02	0.50	0.90
1581	Mean	0.0	0.0	2.7	24.7	0.0	0.0	0.0	3.7	43.7	48.3
	MF	0.00	0.00	0.03	0.27	0.00	0.00	0.00	0.27	0.96	0.97
500	Mean	16.0	13.3	57.0	57.3	5.3	3.7	9.0	14.7	46.7	47.3
	MF	1.00	0.70	0.70	0.63	0.44	0.28	0.71	1.07	1.03	0.95
158.1	Mean	16.7	18.0	66.0	77.3	11.0	8.3	12.3	14.3	49.0	48.0
	MF	1.04	0.95	0.81	0.84	0.92	0.64	0.97	1.05	1.08	0.96
50	Mean	17.0	17.3	87.7	95.0	12.3	9.7	13.3	12.7	47.0	49.0
	MF	1.06	0.91	1.08	1.04	1.03	0.74	1.05	0.93	1.04	0.98
15.81	Mean	16.7	17.7	80.3	98.3	11.7	9.7	12.0	12.3	46.0	49.3
	MF	1.04	0.93	0.99	1.07	0.97	0.74	0.95	0.90	1.01	0.99
5	Mean	16.3	16.7	84.3	94.0	12.0	10.0	12.0	13.0	45.3	47.3
	MF	1.02	0.88	1.04	1.03	1.00	0.77	0.95	0.95	1.00	0.95
1.581	Mean	16.3	17.7	83.0	92.7	12.0	11.3	11.0	12.0	44.0	48.0
	MF	1.02	0.93	1.02	1.01	1.00	0.87	0.87	0.88	0.97	0.96
NPD (4µg)	Mean	408.0	--	--	--	--	--	--	--	--	--
	MF	24.00	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2401.3	--	2485.3	--	225.0	--	223.3	--	--
	MF	--	126.39	--	27.11	--	18.75	--	16.75	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	248.0
	MF	--	--	--	--	--	--	--	--	--	4.83
SAZ (2µg)	Mean	--	--	1221.3	--	1200.0	--	--	--	--	--
	MF	--	--	15.02	--	100.00	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	406.7	--	--	--
	MF	--	--	--	--	--	--	36.97	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1048.0	--
	MF	--	--	--	--	--	--	--	--	23.12	--

 

Table 3: Summary Table of the Assay 2 and Assay 3

Concentrations (µg/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimuriumtester strains								Escherichia coli
		TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	19.3	18.0	105.0	93.7	13.7	12.7	9.7	11.0	45.0	48.3
	MF	1.09	1.04	1.09	0.99	1.05	1.09	0.88	0.97	1.05	0.99
DMSO control	Mean	17.3	17.3	--	91.0	--	11.3	12.7	11.0	--	49.0
	MF	0.98	1.00	--	0.96	--	0.97	1.15	0.97	--	1.01
Distilled water control	Mean	17.7	17.3	96.3	94.7	13.0	11.7	11.0	11.3	42.7	48.7
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	--	0.0	--	8.3	--	0.0	--	0.3	21.3	45.3
	MF	--	0.00	--	0.09	--	0.00	--	0.03	0.50	0.93
1581	Mean	--	0.0	--	4.0	--	7.0	--	7.3	43.3	47.0
	MF	--	0.00	--	0.04	--	0.60	--	0.65	1.02	0.97
500	Mean	4.7	10.7	--	58.0	--	6.0	--	9.7	43.7	48.3
	MF	0.26	0.62	--	0.61	--	0.51	--	0.85	1.02	0.99
158.1	Mean	8.0	15.0	42.3	83.7	5.0	9.7	2.3	11.0	46.0	46.3
	MF	0.45	0.87	0.44	0.88	0.38	0.83	0.21	0.97	1.08	0.95
50	Mean	14.7	17.7	53.3	97.0	12.3	10.0	6.0	9.7	45.7	48.0
	MF	0.83	1.02	0.55	1.02	0.95	0.86	0.55	0.85	1.07	0.99
15.81	Mean	17.3	16.0	103.0	93.3	13.7	12.0	10.7	10.3	45.3	47.0
	MF	0.98	0.92	1.07	0.99	1.05	1.03	0.97	0.91	1.06	0.97
5	Mean	16.0	17.3	107.3	98.0	11.0	11.3	8.3	12.7	46.0	47.0
	MF	0.91	1.00	1.11	1.04	0.85	0.97	0.76	1.12	1.08	0.97
1.581	Mean	17.0	18.3	117.0	99.7	11.0	12.3	12.0	11.7	47.3	45.7
	MF	0.96	1.06	1.21	1.05	0.85	1.06	1.09	1.03	1.11	0.94
0.5	Mean	15.7	--	95.3	--	11.0	--	9.0	--	--	--
	MF	0.89	--	0.99	--	0.85	--	0.82	--	--	--
0.1581	Mean	--	--	117.3	--	13.3	--	--	--	--	--
	MF	--	--	1.22	--	1.03	--	--	--	--	--
NPD (4µg)	Mean	429.3	--	--	--	--	--	--	--	--	--
	MF	24.77	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2294.7	--	2394.7	--	185.0	--	193.3	--	--
	MF	--	132.38	--	26.32	--	16.32	--	17.58	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	242.0
	MF	--	--	--	--	--	--	--	--	--	4.94
SAZ (2µg)	Mean	--	--	1228.0	--	1206.7	--	--	--	--	--
	MF	--	--	12.75	--	92.82	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	420.0	--	--	--
	MF	--	--	--	--	--	--	33.16	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1098.7	--
	MF	--	--	--	--	--	--	--	--	25.75	--

 

Conclusions:

In conclusion, the test item Disodium Lauryl Sulfosuccinate (EC 939-638-8) (Batch Number: 12301) had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Plate Incorporation Method), an Assay 1 (Plate Incorporation Method), an Assay 2 (Pre-Incubation Method) and an Assay 3 (Pre-Incubation Method).

Based on the available information, the stock solution of the test item was prepared in distilled water at a concentration of 100 mg/mL in Assay 1-2, an at 10 mg/mL in Assay 3. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the preliminary experiment, the examined test concentrations in Assay 1 and Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate; based on the observed cytotoxicity in Assay 2 in Salmonella typhimurium bacterial strains without metabolic activation the examined test concentrations in Assay 3 were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.

In the Assay 2 using the pre-incubation method, excessive cytotoxicity was observed in all Salmonella typhimurium tested bacterial strains without metabolic activation at several concentrations. In these cases, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, the experiment in these bacterial strains without metabolic activation was repeated at lower concentrations to complete the data.

No precipitate was detected in any examined bacterial strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item was observed in all Salmonella typhimurium bacterial strains with and/or without metabolic activation in Assays 1-3 at higher concentrations and in Escherichia coli (Escherichia coli WP2 uvrA) bacterial strain without metabolic activation in Assays 1-2 at 5000 μg/plate.

In the assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Disodium Lauryl Sulfosuccinate (EC 939-638-8) (Batch Number: 12301) had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached read-across justification

Reason / purpose for cross-reference:

read-across source

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality:
The pH and osmolality of the negative control and all test item formulations in the medium were determined for each experiment employing the methods given below:
pH values: using a digital pH meter type WTW pH 525 (series no. 51039051),
Osmolality: with a semi-micro osmometer .
No relevant changes in pH or osmolality of the formulations were noted.
- Water solubility:
The test item was completely dissolved in aqua ad iniectabilia.
- Precipitation:
Any possible test item precipitation was checked before and after each experiment. Evaluation of precipitation was done by light microscopy at the beginning and end of treatment.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary experiment without and with metabolic activation test item concentrations of 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL medium were employed. Cytotoxicity was noted starting at a concentration of 156.3 µg test item/mL. Hence, 156.3 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The micronucleus frequencies of the vehicle controls without and with metabolic activation for the last 8 or 7 studies (most recent background data, not audited by the QAU-department) are given as follows:
Micronucleus frequency per 1000 cells
Without metabolic activation (4-h or 20-h exposure):
Untreated control (n = 8):
Mean: 4.9
Standard deviation: 2.0
Range: 1-9
Vehicle control (n = 8):
Mean: 7.2
Standard deviation: 4.6
Range: 1-18
Positive control: Mitomycin C (n = 7):
Mean: 95.8
Standard deviation: 66.1
Range: 24-286
Positive control: Colchicine (n = 7):
Mean: 25.4
Standard deviation: 10.2
Range: 7-43
With metabolic activation (4-h exposure):
Vehicle control (n = 8):
Mean: 10.8
Standard deviation: 6.2
Range: 2-25
Positive control: Cyclophosphamide (n = 7):
Mean: 60.3
Standard deviation: 37.8
Range: 20-147

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, the read-across test item tested up to a cytotoxic concentration of 156.3 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.
In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively.

Executive summary:

Test sample of read-across test item Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts was assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 20 hours after the end of exposure. The study was conducted in duplicate.

The read-across test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. Aqua ad iniectabilia served as the vehicle control.

The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation test item concentrations of 39.1, 78.1, 156.3, 312.5, 625, 1250 and 2500 µg/mL medium were employed. Cytotoxicity was noted starting at a concentration of 156.3 µg test item/mL. Hence, 156.3 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation.

In the main study cytotoxicity was noted at the top concentration of 156.3 µg/mL in the experiments without and with metabolic activation.

Mitomycin C and colchicine were employed as positive controls in the absence and cyclophosphamide in the presence of metabolic activation.

Tests without metabolic activation (4- and 20-hour exposure)

The micronucleus frequencies of cultures treated with the read-across test item at concentrations of 9.77, 19.53, 39.1, 78.1 or 156.3 µg/mL medium (4 h and 20-h exposure) in the absence of metabolic activation ranged from 5.5 to 15.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487; (in this test: vehicle control: 6.0 or 12.0 micronuclei per 1000 binucleated cells, untreated controls: 4.0 or 9.0 micronuclei per 1000 binucleated cells (4-hour and 20-hour exposure, respectively). Vehicle and untreated control values fell within acceptation ranges.

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the read-across test item at concentrations of 9.77, 19.53, 39.1, 78.1 or 156.3 µg/mL medium (4-h exposure) in the presence of metabolic activation ranged from 6.0 to 18.0 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the cytotoxic concentration. Vehicle controls should give reproducibly low and consistent micronuclei frequencies, typically 5 - 25 micronuclei per 1000 cells according to OECD 487; (in this test: vehicle control: 3.5 or 13.0 micronuclei per 1000 binucleated cells, untreated controls: 4.5 or 8.5 micronuclei per 1000 binucleated cells). Vehicle and untreated control values fell within acceptation ranges.

 Under the present test conditions, the read-across test item tested up to a cytotoxic concentration of 156.3 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test.

In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The registered test substance was tested for potential mutagenic activity in a key Bacterial Reverse Mutation Assay in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of a metabolic activation (Varga-Kanizsai, 2022). Based on the results of a preliminary experiment, the examined test concentrations in Assay 1 and Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate; based on the observed cytotoxicity in Assay 2 in Salmonella typhimurium bacterial strains without metabolic activation the examined test concentrations in Assay 3 were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate. In the Assay 2 using the pre-incubation method, excessive cytotoxicity was observed in all S.typhimurium tested strains without metabolic activation at several concentrations. In these cases, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, the experiment in these bacterial strains without metabolic activation was repeated at lower concentrations to complete the data. No precipitate was detected in any examined bacterial strains with and without metabolic activation. Inhibitory, cytotoxic effect of the test item was observed in all S. typhimurium bacterial strains with and/or without metabolic activation in Assays 1-3 at higher concentrations and in Escherichia coli (Escherichia coli WP2 uvrA) bacterial strain without metabolic activation in Assays 1-2 at 5000 μg/plate. In the assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect. The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid. The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the registered test item had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

 

Read-across data were available from analogue substances CAS No. 90268-36-3 (Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts) and CAS 37294-49-8 (Butanedioic acid, sulfo- [C9-11 (branched)alkyl] ester, disodium salts):

Bacterial mutagenicity

- A support study for acute bacterial reverse mutation was available with test item CAS No. 90268-36-3 (Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts) containing > 95% active ingredient in 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (Flügge, 2013c). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. In a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test, ten concentrations of 0.316 up to 5000 µg test item/plate were tested. Cytotoxicity was noted at concentrations of 316 µg/plate and higher. Hence, 316 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test. Six concentrations of 1.0, 3.16, 10.0, 31.6, 100 and 316 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to a cytotoxic concentration of 316 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

Under the present test conditions the test item tested up to a cytotoxic concentration of 316 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

In conclusion, negative results were obtained for mammalian mutagenicity in a key study with read across substance CAS No. 90268-36-3 tested in V79 cells with genetic marker HPRT.

- A supporting Ames test was performed with CAS 37294-49-8 (Butanedioic acid, sulfo- [C9-11 (branched)alkyl] ester, disodium salts) 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.

 

Mammalian mutagenicity

- No data were available for the registered substance, but read across data were available from analogue substance CAS No. 90268-36-3 (Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts), for which a key study was conducted with test item containing >=95% active ingredient in cultured mammalian cells (V79, genetic marker HPRT) both in the presence (4 hours) and absence (4 and 24 hours) of metabolic activation (Flügge, 2013d). Cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 39.06 or 156.3 µg test item/mL without and with metabolic activation (24-h or 4-h exposure), respectively. Hence, concentrations of 2.44, 4.88, 9.77, 19.53 or 39.06 and 9.77, 19.53, 39.06, 78.13 or 156.3 µg test item/mL were selected for the main experiments without and with metabolic activation, respectively. In the main study cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations 39.06 or 156.3 µg/mL in the absence and presence of metabolic activation, respectively. Both in the experiments with and without metabolic activation, the mutation frequencies of treated cell cultures within the normal range of the vehicle controls. The positive controls caused a pronounced increase in the mutation frequencies, indicating the validity of this test system. Under the present test conditions, the test item tested up to cytotoxic concentrations in the experiments without and with metabolic activation, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

In conclusion, negative results were obtained for mammalian mutagenicity in a key study with the read across substance CAS No. 90268-36-3 tested in V79 cells with genetic marker HPRT.

-A supporting in vitro gene mutation assay was conducted with CAS 37294-49-8 (Butanedioic acid, sulfo- [C9-11 (branched)alkyl] ester, disodium salts) 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.

 

Chromosome aberration

- No data were available for the registered substance, but read across data were available from analogue substance CAS No. 90268-36-3 ( Butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts), for which a key study was conducted with test item containing >=95% active ingredient in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (key study; Flügge, 2013e). The test was carried out employing 2 exposure times without S9 mix: 4 and 20 hours, and 1 exposure time with S9 mix: 4 hours. The test item was completely dissolved in aqua ad iniectabilia. A correction factor of 1.05 was used to correct for the purity of the test item. Aqua ad iniectabilia served as the vehicle control. In the preliminary experiment, cytotoxicity was noted starting at a concentration of 156.3 µg test item/mL. Hence, 156.3 µg/mL were employed as the top concentration for the mutagenicity tests without and with metabolic activation. In the main study cytotoxicity was noted at the top concentration of 156.3 µg/mL in the experiments without and with metabolic activation. Both in the experiments with and without metabolic activation, the mutation frequencies of treated cell cultures were within the normal range of the vehicle controls. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Under the present test conditions, the test item tested up to a cytotoxic concentration of 156.3 µg/mL medium, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test. In conclusion, negative results were obtained for chromosome aberration in a key study with read across substance CAS No. 90268-36-3 tested in an in vitro Micronucleus test in human peripheral lymphocytes.

-A supporting in vitro Micronucleus assay was available with CAS 37294-49-8 (Butanedioic acid, sulfo- [C9-11 (branched)alkyl] ester, disodium salts) 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.

Conclusion

Standard information requirements according to REACH Guidance Part 3 R7a were fulfilled for genotoxicity testing, including bacterial and mammalian mutagenicity and chromosomal aberration. Based on the available results, there were no indications of mutagenicity or genotoxicity, and no further testing is needed. The substance can be considered to have no mutagenic or genotoxic potential.

Justification for classification or non-classification

Based on the negative findings with the registered substance and read-across substance, the test item does not need to be classified and has no obligatory labelling requirement for genotoxicity according to CLP (No. 1272/2008 of 16 December 2008).