Naphthalenesulfonic acid, bis(1-methylethyl)-, Me derivs., sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Naphthalenesulfonic acid, bis(1-methylethyl)-,methyl derivs., sodium salt(C7-alkyl naphthalene sulphonate) was not mutagenic in the Salmonella typhimurium reverse mutation assay. Testing in ToxTracker did not lead to induction of the genotoxicity reporters when tested in the absence or presence of a metabolising system (S9).

Link to relevant study records

Reference

Reference 1

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:

2016-10-11 to 2016-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

His

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat S9 liver microsomal fraction

Test concentrations with justification for top dose:

Experiment I:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II:
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: A. dest.

Untreated negative controls:

yes

Remarks:

A. dest., Eurofins Lot No. 160905, 160915

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
-For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

- Preincubation period: 60 min; 100 µL of the test item preparation was pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used. (In a one case only two plates were evaluated)

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

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:

cytotoxicity

Remarks:

experiment I and II

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

Toxic effects of the test item were noted in most tester strains evaluated in experiment I and II.

In experiment I toxic effects of the test item were observed in tester strains TA 98 and TA 1535 at a concentration of 5000 µg/plate and in tester strains TA 100 and TA 1537 at concentrations of 2500 µg/plate and higher (all without metabolic activation).

In experiment II toxic effects of the test item were noted in tester strains TA 98 and TA 1535 at concentrations of 2500 µg/plate and higher (without metabolic activation) and at a concentration of 5000 µg/plate (with metabolic activation). In tester strains TA 100 and TA 1537 toxic effects of the test item were observed at concentrations of 2500 µg/plate and higher (with and without metabolic activation).

Conclusions:

The substance did not induce mutant colonies over background levels in the reverse gene mutation assay.

Executive summary:

In order to investigate the potential of Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

Toxic effects of the test item were noted in most tester strains used in experiment I and II:

- at concentrations of 2500 µg/plate and higher (without metabolic activation), depending on the particular tester strain.

- In experiment II toxic effects of the test item were noted at concentrations of 2500 µg/plate and higher (with and without metabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.

Therefore, Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

genetic toxicity in vitro, other

Remarks:

ToxTracker Assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-09-19 until 2016-10-06

Reliability:

2 (reliable with restrictions)

Qualifier:

no guideline available

Version / remarks:

ToxTracker is a panel of mammalian stem cell lines that contain different fluorescent reporters for induction of DNA damage, oxidative stress and protein damage. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry. The test is under evaluation at OECD for TG.

Principles of method if other than guideline:

ToxTracker consists of a panel of six different mES GFP reporter cell lines representing four distinct biological responses that are associated with carcinogenesis, i.e. general cellular stress, DNA damage, oxidative stress and the unfolded protein response. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry.
The specificity of the ToxTracker reporter cell lines was extensively validated using different libraries of reference compounds as demanded by various regulatory agencies. Over 97% of all tested chemicals were classified correctly, confirming the outstanding performance of the assay. Importantly, compounds that are often erroneously identified as genotoxic compounds by the conventional in vitro tests were identified by the assay as causing predominantly oxidative stress, explaining their positive results.
In comparing sensitivity and specificity of current in vitro genotoxicity assays, it is clear that ToxTracker outperforms regulatory tests as bacterial reversion, chromsomal aberration and mamalian mutation.

Test system Sensitivity(%) Specificity(%)
bacterial reversion (Ames) 60 77
chromsomal aberration 70 55
mamalian mutation 81 48
ToxTracker 95 94
(Walmsley, R.M., and Billinton, N. (2011). British Journal of Pharmacology and Hendriks et al., (2016). Toxicol. Sci.)

GLP compliance:

no

Remarks:

ToxTracker is run in a non-GLP facility. However general principles to conduct proper scientifically correct in vitro experiments were adhered to, and in particular care was taken for proper handling of test compound (stock) solutions.

Type of assay:

other: ToxTracker uses six mammalian stem cell lines with GFP reporters for induction of DNA damage, oxidative stress and protein damage. The induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry.

Target gene:

ToxTracker is a panel of mammalian stem cell lines that contain different fluorescent reporters for induction of DNA damage, oxidative stress and protein damage. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry.

Species / strain / cell type:

other: Mouse embryonic stem cells

Details on mammalian cell type (if applicable):

ToxTracker consists of a panel of six different mES GFP reporter cell lines representing four distinct biological responses that are associated with carcinogenesis, i.e. general cellular stress (Biomarker gene Btg2), DNA damage (Biomarker genes Bscl2 and Rtkn), oxidative stress (Biomarker genes Srxn1 and Blvrb) and the unfolded protein response (Biomarker gene Ddit3).

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

In the screening assay 20 doses with 2-fold dilution steps were tested up to the maximum concentration of 2000 μg/ml.

Vehicle / solvent:

Water

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle controls

Positive controls:

yes

Positive control substance:

other: Cisplatin, Diethyl maleate, Tunicamycin, Aflatoxin B1

Remarks:

Cisplatin with and without S9, Diethyl maleate without S9, Tunicamycin without S9 and Aflatoxin B1 with S9.

Details on test system and experimental conditions:

The assay involves involves the use of 6 mES GFP (Green fluorescent protein) reporter cell lines - each cell-line for a specific biomarker. Dose range finding for cytotoxicity is measured in mES that is not transfected.
GFP reporter induction was always compared to a vehicle control treatment. DMSO concentration was similar in all wells for a particular compound and never exceeds 1%. All compounds have been tested both with and without metabolic activation with S9 in at least three completely independent experiments. Positive reference treatments with cisplatin (DNA damage), diethyl maleate (oxidative stress) and Tunicamycin (unfolded protein response) were included in all assays without S9, and cisplatin and Aflatoxin B1 (DNA damage) were included in all assays with S9.

For chemical testing, the six independent mES reporter cell lines are seeded in gelatin-coated 96-well cell culture plates in 200 μl BRL-conditioned ES cell medium (40.000 cells per well). 24 h after seeding the cells in the 96-well plates, medium is aspirated and fresh BRL-conditioned ES cell medium containing 10% foetal calf serum and the diluted chemicals is added to the cells. For each tested compound, five concentrations are tested in 2-fold dilutions. The highest compound concentration will induce significant cytotoxicity (50-70%). The appropriate dose range for the ToxTracker analysis is determined in a extensive dose range finding. For the dose range finding, as well as the ToxTracker analysis a maximum concentration of 10 mM or 2 mg/ml is used, in line with the advised maximum concentration in the current guidelines for in vitro genotoxicity testing. In case concentrations are limited by solubility or the occurrence of precipitation in the culture medium, the maximum soluble concentration with be used in the assay.
For the dose range finding, wild type mES cells are exposed to 20 different concentrations of the test substances. Cytotoxicity is estimated by cell count after 24 h exposure using a flow cytometer and is expressed as percentage of viable cells after 24 h. exposure compared to unexposed controls. Compounds are diluted in DMSO, water or PBS. For cytotoxicity assessment in the ToxTracker assay, the relative cell survival for the six different reporter cell lines is averaged.
Metabolic activation was included in the ToxTracker assay by addition of S9 liver extract from alachlor-induced rats (Moltox). Cells are exposed to five concentrations of the test compounds in the presence of S9 and required co-factors (RegenSysA+B, Moltox) for 3 h. After washing, cells are incubated for 24 h in fresh BRL-conditioned ES cell medium. Aflatoxin B1 was included as positive control for progenotoxin metabolism.
Induction of the GFP reporters was determined after 24 h exposure using a Guava easyCyte 8HT flow cytometer (Millipore). Only GFP expression in intact single cells was determined. Mean GFP fluorescence and cell concentrations in each well was measured and was used for cytotoxicity assessment.
In case auto-fluorescence of the test substances was observed in the dose range finding, wild type mES cells were exposed to the samples at the same concentrations as used in the ToxTracker. The mean fluorescence caused by the compound was then subtracted from the ToxTracker results of the respective compound.

Evaluation criteria:

The ToxTracker assay was considered to have a positive response when a compound induces at least a 2 fold increase in GFP expression in any of the reporters. Activation of the Bscl2-GFP or Rtkn-GFP reporters indicate induction of DNA damage, Srxn1-GFP and Blvrb-GFP indicated induction of cellular oxidative stress and Ddit3-GFP activation is associated with the unfolded protein response. Only GFP inductions at compound concentrations that showed < 75% cytotoxicity are used for the ToxTracker analysis. Data from measurements > 75% cytotoxicity can not be interpreted in a meaningful way and are therefore discarded.

Statistics:

To compare the induction of the six GFP reporters for a collection of compounds, each with different biological reactivities, dose-response relationships and kinetics, Toxplot calculates for each compound the level of GFP induction for every individual reporter at a specified level of cytotoxicity (typically 10%, 25% and 50%). GFP induction levels are calculated by linear regression between two data points around the specified cytotoxicity level. In case the specified level of cytotoxicity can not be reached at the highest tested compound concentration, Toxplot displays the GFP induction level at this top concentration. In the heatmap, Toxplot clearly marks the compounds that do not induce the selected level of cytotoxicity.
Because the cytotoxicity for a compound can vary between the ToxTracker cell lines, calculations of the GFP induction levels of the individual reporters by Toxplot can slightly deviate from the GFP induction and cytotoxicity figures.

Key result

Species / strain:

other: GFP-based mouse embryonic stem (mES) cell reporter lines

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Cytotoxicity range finder

Cytotoxicity is measured in mES that is not transfected:

• Test in absence and presence of S9 metabolic activation system: 24 h continues exposure in absence of S9; 3 h exposure to the compounds in presence of S9, followed by washing of cells and recovery up to 24 h in absence of the test compounds and S9.

• Solvents: cisplatin in PBS, aflatoxin B1 and test substance in water at 20 2-fold dilutions from 2 mg/mL downward.

• Assay performed following standard protocols. Cytotoxicity is based on viability as measured by cell counts in flow cytometer.

Results: Without S9: No cytotoxicity observed up to 15.6 µg/ml, 100%cytotoxicity from 125 µg/ml.

With S9: No cytotoxicity observed up to 125 µg/ml, 100% cytotoxicity from 250 µg/ml.

Main study:

The full studies, both with and without S9, were run with concentrations 0, 31.3, 62.5, 125, 250, 500 μg/ml. With and without S9, the activation of the different GFP reporters by the control compounds was fully compliant with historical data thereby confirming the technical validity of the performed tests.

 

Without S9:

• 24 h exposure

• Solvents: water, cisplatin in PBS.

• Number of independent tests: 3

• Test in absence of S9 metabolic activation system

• Assay performed following standard protocols

Positive controls: Cisplatin (DNA damage), Diethyl maleate (ROS), Tunicamycin (UPR)

Results: (See attached graphs)

Cytotoxicity: LC50 = 175.1 µg/mL

 

Induction levels of the different ToxTracker reporters at compound concentrations that induce 10%, 25% and 50% cytotoxicity after 24 h exposure.

	DNA damage						Cellular stress			Oxidative stress						Protein damage
	Bscl2			Rtkn			Btg2			Srxn1			Blvrb			Ddit3
Substance	10	25	50	10	25	50	10	25	50	10	25	50	10	25	50	10	25	50
Morwet IP powder	1.0	1.1	1.0	0.9	0.8	0.8	1.0	1.0	0.9	3.2	4.9	3.2	1.1	1.0	0.7	5.7	4.6	2.8
Cisplatin	1.8	3.0	4.2	3.2	5.9	6.6	2.3	3.3	3.6	2.5	3.6	5.3	1.2	1.6	1.7	1.0	1.1	1.2
DEM*	1.3	1.3	1.4	0.9	1.0	1.0	1.6	2.1	2.1	12.9	23.5	23.5	3.8	6.7	7.1	1.3	2.1	2.6
Tunicamycin	1.0	1.0	1.0	0.9	0.8	0.7	0.9	0.9	0.9	1.0	0.8	0.7	0.9	0.8	0.7	3.1	6.3	11.3

Induction factor > 2 in bold

 

With S9:

• Test in presence of S9 metabolic activation system

• 3 h exposure to compound and S9

• 24 h recovery after exposure after washout of compound and S9

• Number of independent tests: 3

• Assay performed following standard protocols

Positive controls: Cisplatin (DNA damage), Aflatoxin B1 (DNA damage)

Results: (See attached graphs)

Cytotoxicity: LC50 = 473 μg/ml

Induction levels of the different ToxTracker reporters at compound concentrations that induce 10%, 25% and 50% cytotoxicity after 3 h exposure in the presence of S9 rat liver extract and 24 h recovery.

	DNA damage						Cellular stress			Oxidative stress						Protein damage
	Bscl2			Rtkn			Btg2			Srxn1			Blvrb			Ddit3
Substance	10	25	50	10	25	50	10	25	50	10	25	50	10	25	50	10	25	50
Morwet IP powder	1.1	1.1	1.1	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.4	1.9	1.1	1.3	1.5	1.1	1.3	1.5
Cisplatin*	3.9	4.3	4.3	6.3	6.3	6.3	2.2	3.7	3.7	2.0	2.9	2.9	1.4	1.9	1.9	0.9	1.0	1.0
Aflatoxin B1*	2.3	3.1	3.3	5.5	6.1	6.3	3.1	3.3	3.5	1.9	3.2	3.8	1.4	1.4	1.4	0.9	0.9	1.0

Induction factor> 2 in bold

* GFP: inductions at the highest tested dose are provided in case the indicated level of cytotoxicity in not reached.

Conclusions:

Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt did not induced the genotoxicity reporters in the ToxTracker assay when tested in the absence or presence of a metabolising system (S9).

Executive summary:

Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt was tested in the ToxTracker assay in the absence and presence of a metabolising system.

ToxTracker consists of a panel of six different mES GFP reporter cell lines representing four distinct biological responses that are associated with carcinogenesis, i.e. general cellular stress, DNA damage, oxidative stress and the unfolded protein response. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry.

The specificity of the ToxTracker reporter cell lines was extensively validated using different libraries of reference compounds as demanded by various regulatory agencies. Over 97% of all tested chemicals were classified correctly. Importantly, compounds that are often erroneously identified as genotoxic compounds by the conventional in vitro tests were identified by the assay as causing predominantly oxidative stress, explaining their positive results. (Hendriks et al, 2016, Toxicol sciences, 150(1) 190-203). The available data on the validation of ToxTracker indicates that it outperforms all currently available regulatory assays on both sensitivity and specificity.

 

The assay involves involves the use of 6 mES GFP (Green fluorescent protein) reporter cell lines - each cell-line for a specific biomarker. Preliminary dose range finding for cytotoxicity was measured in mES that is not transfected.

GFP reporter induction was always compared to a vehicle control treatment. DMSO concentration was similar in all wells for a particular compound and never exceeds 1%. All compounds have been tested both with and without metabolic activation with S9 in at least three completely independent experiments. Positive reference treatments with cisplatin (DNA damage), diethyl maleate (oxidative stress) and Tunicamycin (unfolded protein response) were included in all assays without S9, and cisplatin and Aflatoxin B1 (DNA damage) were included in all assays with S9.

 

Based on observed cytotoxicity in the range finding study, the full studies, both with and without S9, were run with concentrations 0, 31.3, 62.5, 125, 250, 500 μg/ml.

 

Results:

With and without S9, the activation of the different GFP reporters by the control compounds was fully compliant with historical data thereby confirming the technical validity of the performed tests.

The test substance did not show increased cytotoxicity when cells when exposed in presence of S9 rat liver extract, indicating that the substances did not require metabolic activation to become biological reactive. The test substance induced less cytotoxicity when tested in the presence of S9 which is likely caused by a reduced exposure time of 3 h compared to 24 h for exposures in absence of S9.

The test substance when tested with or without S9 showed no activation of tBscl2-GFP reporter, associated with induction of promutagenic DNA lesions that interfered with DNA replication, and no activation of the Rtkn-GFP genotoxicity reporters that are correlated with induction of DNA strand breaks.

There was induction of Nrf2-dependent cellular oxidative stress when exposed in absence of a metabolising system. However, induction levels was 6 to 8-fold lower compared to the positive control Diethyl maleate and was observed at cytotoxic concentrations. In the presence of S9, the oxidative stress was reduced. Most prominent cellular response observed was induction of the unfolded protein response at cytotoxic concentration. Often protein reactivity also results in increased levels of oxidative stress due to reduced function of anti-oxidants like glutathione.

In conclusion, Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt did not lead to induction of reporters indicative for DNA damage. At cytotoxic concentrations increased induction was seen in reporters that are indicative of oxidative stress and unfolded protein.

 

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 studies on genotoxicity.

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 4.2) 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

Ames:

Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt (C7-alkyl naphthalene sulphonate) was tested for itsability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102, both with and without metabolic activation based on phenobarbital and beta-naphthoflavone induced rat S9-mix. The following concentrations were tested in triplicate:

Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment II: 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

No precipitation of the test item was observed in any tester strain used in experiment I and II with and without S9-mix.

Toxicity was observed in most tester strains used in experiment I (without S9) and II (with and without S9) at concentrations of 2500 µg/plate and higher

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

In conclusion,C7-alkyl naphthalene sulphonateis non-mutagenic in the bacterial reverse mutation assay.

 

ToxTracker:

C7-alkyl naphthalene sulphonate was tested in the ToxTracker assay in the presence and absence of a metabolising system. ToxTracker consists of a panel of six different mES GFP reporter cell lines representing four distinct biological responses that are associated with carcinogenesis, i.e. general cellular stress, DNA damage, oxidative stress and the unfolded protein response. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry. GFP reporter induction was always compared to a vehicle control treatment. DMSO concentration was similar in all wells for a particular compound and never exceeds 1%. All compounds have been tested both with and without metabolic activation with S9 in at least three completely independent experiments. Positive reference treatments with cisplatin (DNA damage), diethyl maleate (oxidative stress) and Tunicamycin (unfolded protein response) were included in all assays without S9, and cisplatin and Aflatoxin B1 (DNA damage) were included in all assays with S9.

Based on observed cytotoxicity in the range finding study, the full studies, both with and without S9, were run with concentrations 0, 31.3, 62.5, 125, 250, 500 μg/ml.

Results: With and without S9, the activation of the different GFP reporters by the control compounds was fully compliant with historical data thereby confirming the technical validity of the performed tests.

The test substance did not show increased cytotoxicity when cells when exposed in presence of S9 rat liver extract, indicating that the substances did not require metabolic activation to become biological reactive. The test substance induced less cytotoxicity when tested in the presence of S9 which is likely caused by a reduced exposure time of 3 h compared to 24 h for exposures in absence of S9.

The test substance when tested with or without S9 showed no activation of tBscl2-GFP reporter, associated with induction of promutagenic DNA lesions that interfered with DNA replication, and no activation of the Rtkn-GFP genotoxicity reporters that are correlated with induction of DNA strand breaks.

There was induction of Nrf2-dependent cellular oxidative stress when exposed in absence of a metabolising system. However, induction levels was 6 to 8-fold lower compared to the positive control Diethyl maleate and was only observed at cytotoxic concentrations. In the presence of S9, the oxidative stress was reduced. Most prominent cellular response observed was induction of the unfolded protein response, also at cytotoxic concentration. Often protein reactivity also results in increased levels of oxidative stress due to reduced function of anti-oxidants like glutathione.

In conclusion, C7-alkyl naphthalene sulphonate did not lead to induction of reporters indicative for DNA damage. At cytotoxic concentrations increased induction was seen in reporters that are indicative of oxidative stress and unfolded protein.

 

ToxTracker was extensively validated using different libraries of reference compounds as demanded by various regulatory agencies. Over 97% of all tested chemicals were classified correctly. Importantly, compounds that are often erroneously identified as genotoxic compounds by the conventional in vitro tests were identified by the assay as causing predominantly oxidative stress, explaining their positive results. (Hendriks et al, 2016, Toxicol sciences, 150(1) 190-203). The available data on the validation of ToxTracker indicates that it outperforms all currently available regulatory assays on both sensitivity and specificity.Therefore, additionalin vitromammalian genotoxicity studies to further investigate mutagenic or clastogenic properties are not considered necessary.

 

Supportive information on the non-genotoxic properties ofC7-alkyl naphthalene sulphonateis provided by the available data on the comparable productC10-13-alkyl naphthalene sulphonate, which is different to C7-alkyl naphthalene sulphonate with respect to on average longer alkyl-chain substitution on the naphthalene sulphonate rings: In case of C7-alkyl naphthalene sulphonate, the most common fraction contains substitution of 1 methyl plus 2isopropyl groups, whereas for C10-13-alkyl naphthalene sulfonates, the alkyl substitution mainly consists of one or more methyls and a branched nonene (C9)alkyl group.

Not surprisingly, the structural profiling in QSAR Toolbox (v.4.2) are the same for both substances, and trigger no alerts that are relevant for for genotoxicity. Also physico-chemical properties and toxicological profiles are very comparable.

Finally, when comparing the mechanistic profiling on induction of DNA damage, oxidative stress and/or protein damage as obtained from the series of reporters included in ToxTracker, show a similar profile forC7-alkyl naphthalene sulphonate and C10-13-alkyl naphthalene sulphonate, including a product with lower nonene (C9) levels: i.e. no induction of reporters indicative for DNA damage, and at cytotoxic concentrations and increased induction in reporters that are indicative of oxidative stress and unfolded protein.

 

Further testing ofC10-13-alkyl naphthalene sulphonateconfirmed that it was not mutagenic in the Salmonella typhimurium reverse mutation assay, not clastogenic or aneugenic inin vitromicronucleus assay in cultured peripheral human lymphocytes, and was not mutagenic in thein vitromammalian cell gene mutation test with L5178Y mouse lymphoma cells.

 

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

C7-alkyl naphthalene sulphonatewas not mutagenic in the Salmonella typhimurium reverse mutation assay, and didnot lead to induction of reporters indicative for DNA damage in the ToxTracker assay.

 

Also further property data for sodium alkylnaphthalene sulfonate indicate that genotoxic properties are rather unlikely.

Lack of genotoxicity is further confirmed with cross-reading fromC10-13-alkyl naphthalene sulphonate, which wasnot mutagenic in the Salmonella typhimurium reverse mutation assay, not clastogenic or aneugenic inin vitromicronucleus assay in cultured peripheral human lymphocytes, and was not mutagenic in thein vitromammalian cell gene mutation test with L5178Y mouse lymphoma cells

Therefore no classification for genotoxicity is required.