Reaction mass of sodium (methylbutyl and pentyl) sulfonate and sodium 1,2-bis(pentyloxycarbonyl)ethanesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registered substance (solid fraction) was tested in following in vitro genotoxicity assay: Bacterial Reverse Mutation Assay using Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli (WP2 uvrA) was tested negative up to concentrations of 5000 μg solid fraction of test item/plate in the presence and absence of metabolic activation (Plate incorporation and Pre incubation).

Read-across was performed for genotoxicity endpoints with category members Butanedioic acid, sulfo-, 1,4-bis(1,3-dimethylbutyl)ester, sodium salt (CAS No. 2373-38-8) and Docusate sodium (CAS No. 577-11-7) based on the read-across justification for the Diester Sulfosuccinate Category group.

Based on the experimental data, there was no evidence for bacterial and mammalian gene mutation potential, nor was there any indication of chromosome aberration potential. In conclusion, based on the registered substance and on the read across with experimental data, there is no genotoxic potential for the test substance.

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:

mouse lymphoma L5178Y cells

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

Conclusions:

In conclusion it can be stated that under experimental conditions reported the read-across test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of the read-across test item, Butanedioic acid, sulfo-, 1,4 -bis(1,3 -dimethylbutyl) ester, sodium salt (ca. 80% act. ingr.) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. Both main experiments were performed with and without liver microsomal activation and a treatment period of 4h. The second experiment (experiment IA) was required to verify the results obtained in experiment I and to cover highly toxic concentrations using an adjusted concentration range.

The highest applied concentration in the pre-test on toxicity (5000µg/mL) was chosen with regard to the molecular weight of the read-across test item. The dose range of the main experiments was limited by toxicity of the read-across test item.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was limited by toxicity of the read-across test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Under experimental conditions reported, the read-across test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the read-across test item did not induce structural chromosome aberrations as
determined by the chromosome aberration test in V79 cells ( Chinese hamster cell line) in vitro.
Therefore, Butanedioic acid, sulfo, 1,4-bis(1,3-dimethylbutyl) ester, sodium salt (80% active ingredient) is considered to be non clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.

Executive summary:

The read-across test item Butanedioic acid, sulfo, 1,4-bis(1,3-dimethylbutyl) ester, sodium salt (80% active ingredient), dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments up to 5000 µg/mL(approx. 10 mM of the active ingredient). In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations and 500 cells were scored for polyploidy. Toxic effects indicated by reduced cell numbers and/or mitotic indices of about and below 50% of control were observed in all experimental parts. However, in experiment I in the absence and the presence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. The observed statistical significance's and dose-dependency are regarded as being biologically irrelevant. No increase in the frequencies of polyploid metaphases was found after treatment with the read-across test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the read-across test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the read-across test item is considered to be non-clastagenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 2020 to 16 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", 21 July 1997

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test", 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:

Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse Mutation Test Using Bacteria", 30 May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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: AEROSOL® AY-100 SURFACTANT Batch: WS19C0204 (Solvay)
- Purity, including information on contaminants, isomers, etc.: Active content (Purity): 99.69%
As agreed with the Sponsor, no correction for purity of the test item was applied.

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 the short period of study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Distilled water was used as solvent to prepare the stock solution of the test material. Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent.
- Final concentration of a stock liquid: 100 mg test item /mL stock solution (based on the solubility test)

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:

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:

Target gene:

histidine (Salmonella typhimurium); tryptophan (Eschericha coli)

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Charles River Laboratories Hungary Kft. according to Ames et al. [1] and Maron and Ames [2].
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

Male Wistar rats (444-628 g, animals were 17-20 weeks old and 433-642 g, animals were 13-17 weeks old) 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 were 02 September 2019 and 13 January 2020.
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 were 05 September 2019 and 16 January 2020 (Charles River Laboratories Hungary code: E13142 (it was used in the Preliminary Range Finding Test) and E13222 (it was used in main tests), Expiry date: 05 September 2021 and 16 January 2022).
- method of preparation of S9 mix:
*The S9 Mix (containing 10 % (v/v) of S9):
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 1000.0 mL
Sterilization was performed by filtration through a 0.22 μm membrane filter.
The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate buffer, pH 7.4 500.0 mL
Rat liver homogenate (S9) 100.0 mL
Salt solution for S9 Mix (see above) 400.0 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.
*0.2 M Sodium Phosphate Buffer, pH 7.4
Solution A:
Na2HPO4 x 12 H2O 71.63 g
Distilled water q.s. ad 1000.0 mL
Sterilization was performed at 121oC in an autoclave.
Solution B:
NaH2PO4 x H2O 27.6 g
Distilled water q.s. ad 1000.0 mL
Sterilization was performed at 121oC in an autoclave.
0.2 M 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:
*Assay 1 followed the standard plate incorporation procedure. Bacteria cultured in Nutrient Broth No.2 were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 μL
vehicle or test item formulation (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48(±1) hours.
* Assay 2 followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the Assay 1. Bacteria cultured in Nutrient Broth No.2were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled.
Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48(±1) hours.
- 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 were determined to be 24.5 g/L and 28.0 g/L. 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:

Based on the solubility test, a 100 mg test item /mL stock solution was prepared in Distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and 4 times approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg test item/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Range Finding Test the plate incorporation method was used.
Based on the results of the preliminary test, a 100 mg test item/mL stock solution was prepared in Distilled water, 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 were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate.
Examined concentrations in Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water; DMSO (Dimethyl sulfoxide)

- 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.

- Justification for percentage of solvent in the final culture medium: no

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

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

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two main tests: In Assay 1, the plate incorporation method was used. In Assay 2, the pre-incubation method was used.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation; Assay 1); preincubation (Assay 2)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes at 37°C in a shaking incubator (Assay 2)
- Exposure duration/duration of treatment: at 37°C for 48(±1) hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: In the Assay, 1 no inhibitory, cytotoxic effect of the test item was observed in all bacterial strains with and without metabolic activation.
In Assay 2 inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development) was observed in Salmonella typhimurium TA98, TA100 and TA1537 strains without metabolic activation at 5000 and 1581 μg test item/plate concentrations and with metabolic activation at 5000 μg test item/plate concentration as well as in Salmonella typhimurium TA1535 and Escherichia coli strains without metabolic activation at 5000 μg test item/plate concentration.

METHODS FOR MEASUREMENTS OF GENOTOXICIY: 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.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Evaluation criteria:

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 was 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.

Statistics:

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 Excel software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Assay 2 at 5000 µg test item/plate (no cytotoxicity 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:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Assay 2 at 5000 and 1581 µg test item/plate (no cytotoxicity 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:

in Assay 2 at 5000 µg test item/plate (no cytotoxicity 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:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Assay 2 at 5000 and 1581 µg test item/plate (no cytotoxicity in Assay 1)

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

in Assay 1 and 2

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:

in Assay 2 at 5000 µg test item/plate (no cytotoxicity 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:

in Assay 2 at 5000 µg test item/plate (no cytotoxicity 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:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Assay 2 at 5000 and 1581 µg test item/plate (no cytotoxicity 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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

in Assay 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Assay 2 at 5000 µg test item/plate (no cytotoxicity in Assay 1)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No precipitate was detected on the plates in the preliminary experiment (plate incorporation) in both examined bacterial strains with and without metabolic activation. No precipitate was detected on the plates in the main tests in the all examined bacterial strains with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES (if applicable):
In the Preliminary 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 test item/plate.
No precipitate was detected on the plates in the preliminary experiment in both examined bacterial strains with and without metabolic activation.
No inhibitory or toxic effects of the test item was observed in the preliminary experiment in both examined bacterial strains with and without metabolic activation.
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.

Ames test:
- Signs of toxicity: No inhibitory, cytotoxic effect of the test item was observed in Assay 1. In Assay 2 inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development) was observed in Salmonella typhimurium TA98, TA100 and TA1537 strains without metabolic activation at 5000 and 1581 µg test item/plate concentrations and with metabolic activation at 5000 µg test item/plate concentration as well as in Salmonella typhimurium TA1535 and Escherichia coli strains without metabolic activation at 5000 μg test item/plate concentration.

Table 1:Summary Table of the Assay 1

Concentrations (µg test item/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	19.7	21.0	82.3	93.3	14.7	14.7	14.7	11.7	44.3	45.7
	MF	1.00	0.90	1.02	1.03	1.00	0.98	1.26	0.97	1.02	0.99
DMSO control	Mean	19.3	22.3	--	92.0	--	13.3	11.3	11.0	--	45.3
	MF	0.98	0.96	--	1.01	--	0.89	0.97	0.92	--	0.99
Distilled water control	Mean	19.7	23.3	80.7	90.7	14.7	15.0	11.7	12.0	43.3	46.0
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	17.0	22.3	63.3	78.0	16.7	14.3	10.3	12.3	40.3	45.0
	MF	0.86	0.96	0.79	0.86	1.14	0.96	0.89	1.03	0.93	0.98
1581	Mean	16.7	20.0	75.3	86.3	14.0	15.7	11.0	12.0	39.0	44.3
	MF	0.85	0.86	0.93	0.95	0.95	1.04	0.94	1.00	0.90	0.96
500	Mean	17.0	22.7	77.0	85.0	16.0	17.3	11.3	13.0	42.0	45.0
	MF	0.86	0.97	0.95	0.94	1.09	1.16	0.97	1.08	0.97	0.98
158.1	Mean	16.0	21.3	79.3	91.3	15.3	17.0	11.3	12.0	42.3	44.3
	MF	0.81	0.91	0.98	1.01	1.05	1.13	0.97	1.00	0.98	0.96
50	Mean	16.0	20.0	83.3	97.0	16.3	15.7	10.0	11.7	43.3	46.0
	MF	0.81	0.86	1.03	1.07	1.11	1.04	0.86	0.97	1.00	1.00
15.81	Mean	16.7	18.0	83.7	92.0	16.7	16.0	12.0	13.7	42.0	46.0
	MF	0.85	0.77	1.04	1.01	1.14	1.07	1.03	1.14	0.97	1.00
NPD (4µg)	Mean	428.0	--	--	--	--	--	--	--	--	--
	MF	22.14	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2456.0	--	2473.3	--	215.3	--	208.0	--	--
	MF	--	109.97	--	26.88	--	16.15	--	18.91	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	254.0
	MF	--	--	--	--	--	--	--	--	--	5.60
SAZ (2µg)	Mean	--	--	1057.3	--	1217.3	--	--	--	--	--
	MF	--	--	13.11	--	83.00	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	429.3	--	--	--
	MF	--	--	--	--	--	--	37.88	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1093.3	--
	MF	--	--	--	--	--	--	--	--	25.23	--

Table 9:Summary Table of the Assay 2

Concentrations (µg test item/plate)	Mean values of revertants / Mutation factor (MF)	Salmonella typhimurium tester strains								Escherichia coli
		TA98		TA100		TA1535		TA1537		WP2 uvrA
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	Mean	18.7	22.7	94.0	99.0	16.3	12.7	12.7	12.3	43.3	46.7
	MF	1.00	1.01	1.06	0.94	1.23	0.97	1.41	1.00	0.98	1.01
DMSO control	Mean	17.3	20.3	--	105.0	--	13.3	11.7	13.3	--	45.3
	MF	0.93	0.91	--	1.00	--	1.03	1.30	1.08	--	0.98
Distilled water control	Mean	18.7	22.3	89.0	105.0	13.3	13.0	9.0	12.3	44.0	46.3
	MF	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
5000	Mean	3.7	18.0	0.0	75.3	4.7	13.7	4.0	4.7	11.7	41.3
	MF	0.20	0.81	0.00	0.72	0.35	1.05	0.44	0.38	0.27	0.89
1581	Mean	17.3	19.3	67.3	102.0	11.3	12.0	2.0	13.0	40.3	47.3
	MF	0.93	0.87	0.76	0.97	0.85	0.92	0.22	1.05	0.92	1.02
500	Mean	17.0	19.3	84.7	98.0	15.3	14.3	10.7	13.0	41.0	48.0
	MF	0.91	0.87	0.95	0.93	1.15	1.10	1.19	1.05	0.93	1.04
158.1	Mean	17.0	19.0	90.0	102.3	15.7	14.0	11.7	12.3	42.7	44.7
	MF	0.91	0.85	1.01	0.97	1.18	1.08	1.30	1.00	0.97	0.96
50	Mean	15.7	20.0	90.0	101.7	15.0	14.3	10.7	10.3	43.0	48.3
	MF	0.84	0.90	1.01	0.97	1.13	1.10	1.19	0.84	0.98	1.04
15.81	Mean	17.0	18.7	92.7	109.7	14.7	14.3	11.0	12.7	42.0	47.7
	MF	0.91	0.84	1.04	1.04	1.10	1.10	1.22	1.03	0.95	1.03
5	Mean	15.7	18.3	85.7	105.0	13.7	14.0	10.7	12.3	44.0	47.3
	MF	0.84	0.82	0.96	1.00	1.03	1.08	1.19	1.00	1.00	1.02
NPD (4µg)	Mean	410.7	--	--	--	--	--	--	--	--	--
	MF	23.69	--	--	--	--	--	--	--	--	--
2AA (2µg)	Mean	--	2453.3	--	2449.3	--	218.3	--	234.0	--	--
	MF	--	120.66	--	23.33	--	16.38	--	17.55	--	--
2AA (50µg)	Mean	--	--	--	--	--	--	--	--	--	246.0
	MF	--	--	--	--	--	--	--	--	--	5.43
SAZ (2µg)	Mean	--	--	1138.7	--	1189.3	--	--	--	--	--
	MF	--	--	12.79	--	89.20	--	--	--	--	--
9AA (50µg)	Mean	--	--	--	--	--	--	412.0	--	--	--
	MF	--	--	--	--	--	--	35.31	--	--	--
MMS (2µL)	Mean	--	--	--	--	--	--	--	--	1060.0	--
	MF	--	--	--	--	--	--	--	--	24.09	--

 

Conclusions:

In conclusion, the test item AEROSOL® AY-100 SURFACTANT (Batch Number: WS19C0204) 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-naphthoflavoneinduced rats.

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

Based on the results of the Compatibility Test, the test item was dissolved in Distilled water. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg test item/plate were examined in the 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 the Assay 1 were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate and in the Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item /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.

No precipitate was detected on the plates in the main tests in all examined Bacterial strains with and without metabolic activation.

No inhibitory, cytotoxic effect of the test item was observed in Assay 1.

In Assay 2 inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development) was observed in Salmonella typhimurium TA98, TA100 and TA1537 strains without metabolic activation at 5000 and 1581 μg test item/plate concentrations and with metabolic activation at 5000 μg test item/plate concentration as well as in Salmonella typhimurium TA1535 and Escherichia coli strains without metabolic activation at 5000 μg test item/plate concentration. 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 AEROSOL® AY-100 SURFACTANT (Batch Number: WS19C0204) had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial mutation

A key Bacterial Reverse Mutation Assay according to OECD Test Guidance No. 471  was performed for the registered substance using Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli (WP2 uvrA) in the presence and absence of metabolic activation. The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test, an Assay 1 (Plate Incorporation Method) and an Assay 2 (Pre-Incubation Method). Based on the results of the Compatibility Test, the test item was dissolved in Distilled water. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg test item/plate were examined in the 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 the Assay 1 were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate and in the Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item /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. No precipitate was detected on the plates in the main tests in all examined Bacterial strains with and without metabolic activation. No inhibitory, cytotoxic effect of the test item was observed in Assay 1. In Assay 2 inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development) was observed in Salmonella typhimurium TA98, TA100 and TA1537 strains without metabolic activation at 5000 and 1581 μg test item/plate concentrations and with metabolic activation at 5000 μg test item/plate concentration as well as in Salmonella typhimurium TA1535 and Escherichia coli strains without metabolic activation at 5000 μg test item/plate concentration. 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 EC 941-224-7 had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study (Orovecz, 2021).

Read-across:

- In a supporting Ames test (Flügge, 2013) Butanedioic acid, sulfo-, 1,4-bis(1,3-dimethylbutyl) ester, sodium salt (CAS No. 2373-38-8) was examined in the 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation up to concentration of 5000 µg/plate. The first experiment was carried out as a plate incorporation test and the second as a preincubation test. No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test. No increase in revertant colony numbers as compared with control counts was observed for the test item up to 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively.

- In a supporting study, Docusate sodium (CAS No. 577-11-7) was tested in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 (Clare 1993). After a range-finder experiment showing cytotoxicity at the highest concentration of 5000 µg/plate, maximum test concentrations of 1000 and 2500 µg/plate were chosen for the main experiment 1. In experiment 1, concentrations were close to the limit of toxicity, therefore for experiment 2, concentrations for all strains were maximally 2000 µg/plate without S9 and 2500 µg/plate with S9. In both experiments, Docusate sodium did not result in statistically significant increases in revertant number of colonies, both with and without S9.

Mammalian mutagenicity

Read-across:

- A key study was available for the mutation assay at the thymidine kinase locus (TK+/-) in Mouse lymphoma L5178Y cells for read across substance CAS No. 2373-38-8 according to OECD 476 and GLP guidelines, and was considered to be reliable, adequate and relevant (Wollny, 2006). The assay was performed in two independent experiments, using two parallel cultures each. Both main experiments were performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment (experiment IA) was required to verify the results obtained in experiment I and to cover highly toxic concentrations using an adjusted concentration range. The highest applied concentration in the pre-test on toxicity (5000 μg/mL) was chosen with regard to the molecular weight of the test item. The dose range of the main experiments was limited by toxicity of the test item. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item. In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Chromosome aberration

Read-across

- A key study for chromosome aberration potential in Chinese Hamster V79 cells was available for a read across substance CAS No. 2373-38-8 from the same category (Schulz, 2003). The study was conducted according to OECD 473 and GLP guidelines, and was considered to be reliable, adequate and relevant. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction. A pre-test for cytotoxicity testing was performed up to 5000 µg/mL, resulting in reduced cell numbers after 4h treatment with 625 µg/mL and above in the absence and the presence of S-9 mix. Considering the toxicity data of the pre-test, 800 µg/mL (without S9) and 1000 µg/mL (with S-9) were chosen as top concentrations in the main experiment I. Dose selection of experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24h exposure with 312,5 µg/mL and above. Therefore 600 µg/mL was chosen as top treatment concentration for continuous exposure in the absence of S-9 and 800 µg/mL in the presence of S-9.In both experiments, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed after treatment with the test item. No increase in frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate positive controls induced statistically significant increases in cells with structural chromosome aberrations. In conclusion, the test material was considered to be non clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.

Conclusion

Standard information requirements according to REACH Guidance Part 3 R7a were fulfilled for genotoxicity testing, including bacterial mutagenicity (registered and read-across substances) and mammalian mutagenicity and chromosomal aberration (read-across substances). 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.

Further information supporting the safety of the test substance is provided in the read-across justification (read-across substances: Docusate sodium (CAS No. 577-11-7) and Butanedioic acid, sulfo-, 1,4-bis(1,3-dimethylbutyl) ester, sodium salt (CAS No. 2373 -38 -8)) for the Diester category, showing that the group was negative in vitro for bacterial/mammalian mutagenicity and chromosomal aberration and in vivo for micronuclei formation   (justification with data matrix separately attached in Section 13).

Justification for classification or non-classification

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