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Genetic toxicity in vitro

Description of key information

In a key Ames test the test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation. In a key Chromosome aberration toxicity study in V79 cell of the Chinese hamster in vitro, no structural chromosome and numerical aberrations were observed with and without metabolic activation mix when tested up to cytotoxic concentrations. In a key Mouse Lymphoma assay in the cell line L5178Y, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay in the absence and presence of metabolic activation when tested up to cytotoxic concentrations.

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10% fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Experiment 1 (-S-9): 50, 100, 200, 400, 600 and 800 µg/mL
Experiment 1 (+S-9): 100, 200, 400, 600, 800 and 1000 µg/mL
Experiment 2 (–S-9): 50, 100, 200, 300, 400 and 600 µg/mL
Experiment 2 later sampling time (–S-9): 200, 300, 400 and 600 µg/mL
Experiment 2 (+S-9): 50, 100, 200, 400, 600 and 800 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to cell cultures
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
yes
Remarks:
culture medium
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
in the -S-9 group
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
yes
Remarks:
culture medium
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in the +S-9 group
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Without S9:Exp1: 4h; Exp2: 18h and 28 h; With S9: Exp1 : 4h; Exp2: 4 h
- Expression time (cells in growth medium): 14 h for Exp1 (-S9) and Exp2 (+ and - S9)
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h (Exp 1 and 2), 28 h (Exp2)

SELECTION AGENT (mutation assays): Not applicable
SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 100/concentration (500/concentration for polyploidy)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes


Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0-4.0% aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0-4.0% aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of number of structural chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher’s exact test (p<0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:

A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0-8.5% polyploid cells).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect
- Effects of osmolality:no effect
- Evaporation from medium: not applicable
- Water solubility: good soluble
- Precipitation: not
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: not applicable
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 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 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 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 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 test item is considered to be non-clastagenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
The acceptability criteria of the positive controls were augmented according to the latest IWGT recommendations. This deviation had no detrimental impact on the outcome of the study.
Principles of method if other than guideline:
The acceptability criteria of the positive controls were augmented according to the latest IWGT recommendations.
This deviation had no detrimental impact on the outcome of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
mouse lymphoma thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: HAT & RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: thymidine Kinase TK+/-
Metabolic activation:
with and without
Metabolic activation system:
S-9
Test concentrations with justification for top dose:
Experiment I
without S-9: 18.8; 37.5; 75.0; 112.5; and 150.0 µg/mL
with S-9: 18.8; 37.5; 75.0; 150.0; 225.0 µg/mL

Experiment IA
without S-9: 125.0; 150.0; 175.0; and 200.0 µg/mL
with S-9: 200.0; 225.0; 250.0; 275.0; and 300.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: highly soluble
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
yes
Remarks:
culture medium
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: without S-9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
yes
Remarks:
culture medium
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: with S-9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension in medium

DURATION
- Preincubation period: 3 days (1 day in RPMI-HAT medium and 2 days recovery in RPMI 1640 medium)
- Exposure duration: Experiment I and Experiment IA with and without S-9 =4 hours
- Expression time (cells in growth medium):48 hours
- Selection time (if incubation with a selection agent):10-15 days
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth(RTG);Relative suspension growth (RSG)
Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10+6 cells above the corresponding solvent control.

A relevant increase of the mutation frequency should be dose-dependent.

A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.

However, in the evaluation of the test results the historical variability of the mutation rates in negative and vehicle controls and the mutation rates of all negative and vehicle controls of this study are taken into consideration.

Results of test groups are rejected if the relative total growth, and the cloning efficiency 1 is less than
10 % of the vehicle control.
Statistics:
A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance should be considered together.
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 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 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 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 limited by toxicity of the 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 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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S2(R1 ): Guidance on Genotoxicity Testing and Data lnterpretation for Pharmaceuticals lntended for Human Use, Current Step 4 version dated November 9, 2011.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary test: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg/plate
Main tests : 31.6, 100, 316, 1000, 3160 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectabilia
- Justification for choice of solvent/vehicle: The test item was completely dissolved in aqua ad iniectabilia .
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide in aqua ad iniectabilia
Remarks:
TA 1535, TA 100 (10 µg/plate): without S9-mix
Positive controls:
yes
Positive control substance:
other: 2-Nitro-fluorene in DMSO
Remarks:
TA 98 ( 10 µg/plate): without S9-mix
Positive controls:
yes
Positive control substance:
other: 9-Amino-acridine in ethanol, abs.
Remarks:
TA 1537 ( 100 µg/plate): without S9-mix
Positive controls:
yes
Positive control substance:
other: Mitomycin C in DMSO
Remarks:
TA 102 ( 10 µg/plate): without S9-mix
Positive controls:
yes
Positive control substance:
other: Benzo(a)pyrene in DMSO
Remarks:
TA 98, TA 102, TA 1537 ( 10 µg/plate): with S9-mix
Positive controls:
yes
Positive control substance:
other: 2-amino-anthracene in DMSO
Remarks:
TA 100, TA 1535 (2 µg/plate): with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation): Initial mutagenicity assay
preincubation: Confirmatory mutagenicity assay

DURATION
1 st independent experiment
- Plate lncorporation Method
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours
2nd independent experiment - Preincubation Method
- Preincubation period: 20 min
- Exposure duration: 48 to 72 hours
- Selection time (if incubation with a selection agent): 48 to 72 hours

SELECTION AGENT (mutation assays): histidine (a sterile solution of 0.5 mM L-histidine HCl/0.5 mM biotin)

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants, a clearing or diminution of the background lawn, or by the degree of survival of the treated cultures




Evaluation criteria:
The statistical evaluation of the results of the AMES test is still under discussion. In our laboratory, a test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared with the vehicle control to at least 2-fold of the vehicle control for TA98, TA100 and TA102 and 3-fold of the vehicle control for TA1535 and TA1537 in both independent experiments;
Or
- a concentration-related increase of the revertants is observed. The Spearman's rank correlation coefficient may be applied.
Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in all test strains.
Vehicle controls validity:
valid
Untreated negative controls validity:
other: vehicle controls valid; no true negaitve controls performed
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 with metabolic activation
negative without metabolic activation

Under the present test conditions the test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
Executive summary:

The test item 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 (a microsomal preparation derived from Aroclor 1254-induced rat liver). 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. Aqua ad iniectabilia was used as vehicle control.

Preliminary test

The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA100 employing a plate incorporation test. Ten concentrations of 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations of 31.6, 100, 316, 1000, 3160 and 5000 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in all test strains.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The results for the vehicle controls were within the range of historical control data of the laboratory. The positive control items showed a significant increase in the number of revertant colonies compared to the vehicle controls of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

In conclusion, under the present test conditions the test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

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

Additional information

Bacterial mutagenicity

In a key Ames test (Flügge, 2013) the test item 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. 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. Aqua ad iniectabilia was used as vehicle control. Based on a preliminary cytotoxicity test, six concentrations of 31.6, 100, 316, 1000, 3160 and 5000 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 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, tested up to a concentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

Mammalian gene mutation

In a key Mouse Lymphoma assay in the cell line L5178Y(Wollny, 2006), a test item containing 80% active ingredient was assessed for its potential to induce mutations in the mouse lymphoma thymidine kinase locus using the cell line L5178Y with and without liver microsomal activation and a treatment period of 4h. After a dose range finding study, two independent experiments with in duplo cell cultures were tested up to highly toxic concentrations leading to 10 -20% viability. 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 test item. Under the experimental conditions reported, the test substance 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

In a key Chromosome aberration toxicity study in V79 cell of the Chinese hamster in vitro (Schulz, 2003), a test item containing 80% active ingredient was assessed for its potential to induce structural chromosome aberrations in two independent experiments up to 5000 µg act.ingr./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. 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 and numerical chromosomal aberrations was observed after treatment with the test item. Appropriate mutagens were used as positive controls.

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. The substance can be considered to have no mutagenic or genotoxic potential.

An in vivo cytogenetics assay was requested based on ECHA Communication number CCH-D-2114330559-45-01/F. The results will be provided as soon as possible with an update of the dossier.Docusate sodium was used as read across substance for the registration of potassium 1,2-bis(2-ethylhexyloxycarbonyl)ethanesulphonate (CAS 7491-09-0). Based on the results of the in vitro chromosomal abberation (OECD 473 -) study wiht docusate sodium, which showed increases in the proportion of cells with structural aberrations, ECHA requested an in vivo cytogenetics assay (mammalian erythrocyte micronucleus test, mammalian bone marrow chromosomal aberration test or mammalian alkaline comet assay). The new study with docusate sodium will be conducted for both the registrations of bothpotassium 1,2-bis(2-ethylhexyloxycarbonyl)ethanesulphonate and docusate sodium, but also for the other diester sulfosuccinates.


Justification for classification or non-classification

Based on these results and according to the EC Directive (No.93/21/EEC) and CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for genetic toxicity.