Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Sodium alkylnaphthalene sulfonate was not mutagenic in the Salmonella typhimurium reverse mutation assay, not clastogenic or aneugenic in in vitro micronucleus assay in cultured peripheral human lymphocytes, and was not mutagenic in the in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31-Oct-2011 to 10-Nov-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Test compound was soluble in water and water has been accepted and approved by authorities and international guidelines

A homogeneous suspension could be in DMSO and DMSO is accepted and approved by authorities and international guidelines

Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive control substance:
2-nitrofluorene
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
sodium azide
Remarks:
without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
but tested up to the recommended dose level of 5000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
but tested up to the recommended dose level of 5000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate
Conclusions:
Interpretation of results (migrated information):
negative

Sodium alkylnaphthalene sulfonate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Sodium alkylnaphthalene sulfonate did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10-Oct-2011 to 07-Apr-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human peripheral blood
Details on mammalian cell type (if applicable):
Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 3 and 24 exposure: 53, 176, 529, 1762 and 2646 µg/mL
With S9-mix, 3 exposure: 53, 176, 529, 1762 and 2646 µg/mL
First cytogenetic test :
Without and with S9-mix, 3hr exposure; 27 hr fixation: 529, 1058 and 1323 µg/mL
Additional cytogenetic test :
Without and with S9-mix, 4.5hr exposure; 27 hr fixation: 529, 1058 and 1190 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 53, 476 and 582 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle:
Test compound was stable in water and soluble in culture medium. Culture medium has been accepted and approved by authorities and international guidelines

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 Migrated to IUCLID6: MMC-C 0.25 µg/mL for a 3 hours exposure period and 0.15 µg/mL for a 24 hours exposure period
Positive control substance:
other: colchicine: 0.1 µg/ml for a 3 hours exposure period and 0.05 µg/ml for a 24 hours exposure period
Remarks:
without S9
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: 15 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)
Evaluation criteria:
A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.
A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.
Statistics:
The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics:
Key result
Species / strain:
lymphocytes: human peripheral blood
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:
Solvent control: 7.4
2646 µg/ml: 7.4
- Effects of osmolality: No
Solvent control: 0.294 mOsm/kg
2646 µg/ml: 0.328 mOsm/kg


- Precipitation: No precipitation was observed up to and including the top dose of 2646 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 1762 µg/ml and above in the absence and presence of S9, 3 hr treatment/24 hr fixation ; at the dose level of 2646 µg/ml in the absence of S9 for the continuous treatment of 24 hr.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.





 In the first cytogenetic assay in the cultures treated with 1058, 1190 and 1323 µg/ml Sodium alkylnaphthalene sulfonate not enough mono- and binucleated cells could be scored for micronuclei in both cultures. Due to high cytotoxicity a low number of mono- and binucleated cells was present on the slides and therefore not enough cells could be examined for micronuclei. Since two repeat experiments were performed, 6 concentrations were scored for the number of micronuclei. Only three concentrations are recommended according to OECD 487. Therefore this deviation does not influence the study integrity.

Conclusions:
Interpretation of results (migrated information):
negative

Sodium alkylnaphthalene sulfonate is not clastogenic or aneugenic in human lymphocytes
Executive summary:

The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the first cytogenetic assay. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Sodium alkylnaphthalene sulfonate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the independently repeated experiments.

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:
05-Sep-2011 to 29-Nov-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test 1:
Without and with S9-mix, 3 hours treatment: 100, 333, 1000, 3330 and 5000 µg/mL
Without S9-mix, 24 hours treatment: 0.5, 5.3, 26, 53 and 106 µg/ml
Dose range finding test 2:
Without S9-mix, 3 and 24 hours treatment: 50, 150, 250, 325, 400, 500 and 600 µg/mL
Experiment 1:
Without S9-mix, 3 hours treatment: 1, 10, 50, 100, 150, 175, 200 and 225 µg/mL
With S9-mix, 3 hours treatment: 0.5, 5, 53, 159, 264, 370, 423 and 476 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 10, 150, 175, 200, 225, 250, 275 and 300 µg/mL
With S9-mix, 3 hours treatment: 100, 200, 250, 300, 400, 450, 500 and 550 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium (RPMI 1640 medium (Hepes buffered medium (Dutch modification))
- Justification for choice of solvent/vehicle: Test compound was soluble in culture medium and this has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9

Migrated to IUCLID6: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9

Migrated to IUCLID6: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
Solvent control: 7.4
5000 µg/ml: 7.4
- Effects of osmolality: No
Solvent control: 0.294 mOsm/kg
5000 µg/ml: 0.328 mOsm/kg

- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 250 µg/mL and above in the absence of S9, 3 hours treatment; at dose levels of 333 µg/mL and above in the presence of S9, 3 hours treatment; at dose levels of 250 µg/mL and above in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 71 and 89% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period in the first and second experiment, respectively.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 90 and 81% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.
Remarks on result:
other: strain/cell type: Test system L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.

A correction factor of 1.89 was used to correct for the purity.

Conclusions:
Interpretation of results (migrated information):
negative

The mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles.
Sodium alkylnaphthalene sulfonate is not mutagenic in the mouse lymphoma L5178Y test system.
Executive summary:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range

 

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, Sodium alkylnaphthalene sulfonate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

 

In the presence of S9-mix, Sodium alkylnaphthalene sulfonate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

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

Genetic toxicity in vivo

Description of key information

There are no data available from in vivo mutagenicity studies.

Mode of Action Analysis / Human Relevance Framework

Structure profile supports the lack of DNA binding, and none of the relevant profilers for genotoxicityin QSAR Toolbox (v 3.4) triggered a concern.

Based on its surfactant properties, the structure is not expected to easily pass membrane structures. Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and subsequent the nucleus to interact with DNA.

Additional information

For each endpoint bacterial mutagenicity, mammalian mutagenicity and mammalian clastogenicity one study is available on the basis of testing with C10-13 aromatic hydrocarbons reaction products with branched nonene, sulfonated sodium salts. These studies indicate that the substance does not possess mutagenic, clastogenic or aneugenic properties.

 

Lack of genotoxic potential is further supported by structure profiling indicating a lack of DNA binding. Further, none of the relevant profilers for genotoxicityin QSAR Toolbox (v 3.4) triggered a concern.

Based on its surfactant properties, the structure is not expected to easily pass membrane structures. Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and subsequent the nucleus to interact with DNA.

Justification for classification or non-classification

Sodium alkylnaphthalene sulfonate was not mutagenic in the Salmonella typhimurium reverse mutation assay, not clastogenic or aneugenic in in vitro micronucleus assay in cultured peripheral human lymphocytes, and was not mutagenic in the in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. Also further property data for sodium alkylnaphthalene sulfonate indicate that genotoxic properties are rather unlikely.

Therefore no classification for genotoxicity is required