Naphthalene sulfonic acid, reaction products with isobutanol,sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data is available on the product Sodium diisobutylnaphthalenesulphonate (ANS DIB; C8-alkyl naphthalene sulfonate) itself. Evaluation is based on read-across to the comparable C7-alkyl naphthalene sulfonate (ANS IP) and other substances in the Alkyl Naphthalene Sulfonates (ANS) category.

ANS N (‘high nonene’) and ANS IP were not mutagenic in the Salmonella typhimurium reverse mutation assay. ANS N (‘high nonene’) was further 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.

In addition, ANS IP, ANS N (‘high nonene’) and ANS N (‘low nonene’) were tested in the ToxTracker assay in the presence and absence of a metabolising system. The results are similar for all three substances, showing no induction of reporters indicative for DNA damage, and at cytotoxic concentrations an increased induction in reporters that are indicative of oxidative stress and unfolded protein.

 

Structure profiles support the lack of DNA binding, and none of the relevant profilers for genotoxicity in 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.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vitro, other

Remarks:

ToxTracker Assay

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

2016-09-19 until 2016-10-06

Reliability:

2 (reliable with restrictions)

Justification for type of information:

See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

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.

Specific details on test material used for the study:

PETRO AG Special powder
Identification: Sodium akylnaphthalene sulfonate (low nonene)
Chemical name: Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated sodium salts, (low nonene substitution)
Structure: UVCB
Molecular formula: UVCB
Molecular weight: UVCB
CAS Number: 1258274-08-6
Description: Off-white powder
Purity 96.7 w/w%
Test substance storage At room temperature in the dark under nitrogen
Stable under storage conditions until 20 February 2018 (expiry date)

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 approx. 100 µg/ml, 100%cytotoxicity from 500 µg/ml.

With S9: No cytotoxicity observed up to 500 µg/ml, 100% cytotoxicity from 1000 µg/ml.

Main study:

The full studies were run with concentrations 0, 62.5, 125, 250, 500, 1000 μg/mL without S9, and 0, 125, 250, 500, 1000, 2000 μg/mg/mL with S9.

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 = 410.9 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
Petro AG special powder	1.0	1.0	1.0	1.0	0.9	0.8	1.1	1.0	1.0	1.5	2.3	3.6	1.5	1.7	1.9	1.5	3.1	5.6
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 = 1635.2 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 LC	10	25	50	10	25	50	10	25	50	10	25	50	10	25	50	10	25	50
Petro AG special powder	0.9	0.9	0.7	1.0	0.9	0.8	1.0	0.9	0.8	1.0	1.0	1.0	1.1	1.0	0.8	1.0	1.0	1.0
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:

Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulfonated, sodium salts (low nonene) did not induced the genotoxicity reporters in the ToxTracker assay when tested in the absence or presence of a metabolising system (S9).

Executive summary:

Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulfonated, sodium salts (low nonene) was tested in the ToxTracker assay in the 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.

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 were run with concentrations 0, 62.5, 125, 250, 500, 1000 μg/mL without S9, and 0, 125, 250, 500, 1000, 2000 μg/mg/mL with S9.

 

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, Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulfonated, sodium salts (low nonene) did not lead to induction of reporters indicative for DNA damage. At cytoxic concentrations incerased induction was seen in reporters that are indicative of oxicdative strress and unfolded protein.

 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting 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.

Justification for type of information:

See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

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

GLP compliance:

yes (incl. QA statement)

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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting 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.

Justification for type of information:

See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting 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.

Justification for type of information:

See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

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. QA statement)

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.

Reference 5

Endpoint:

genetic toxicity in vitro, other

Remarks:

ToxTracker Assay

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

2016-09-19 until 2016-10-06

Reliability:

2 (reliable with restrictions)

Justification for type of information:

See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

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.

Specific details on test material used for the study:

Trade name: Petro S

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 31 µg/ml, 100% cytotoxicity from 500 µg/ml.

With S9: No cytotoxicity observed up to 500 µg/ml, 100% cytotoxicity from 1000 µg/ml.

Main study:

The full studies were run with concentrations 0, 7.8, 15.6, 31.3, 62.5, 125 μg/mL without S9, and 0, 62.5, 125, 250, 500, 1000 μg/mg/mL with S9.

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 = 119.6 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
Petro S powder	1.0	1.0	1.1	1.0	1.0	0.8	1.0	1.0	0.9	1.0	2.1	3.4	1.0	1.0	1.0	1.7	4.9	10.2
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 = 386.3 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
Petro AG special powder	1.0	1.0	1.0	1.0	1.0	1.0	1.0	1.0	0.9	1.0	1.0	0.9	1.1	1.0	1.0	1.0	1.0	0.9
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:

Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulfonated, sodium salts did not induced the genotoxicity reporters in the ToxTracker assay when tested in the absence or presence of a metabolising system (S9).

Executive summary:

Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulfonated, sodium salts was tested in the ToxTracker assay in the 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.

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 were run with concentrations 0, 7.8, 15.6, 31.3, 62.5, 125 μg/mL without S9, and 0, 62.5, 125, 250, 500, 1000 μg/mg/mL with S9.

 

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, Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulfonated, sodium salts did not lead to induction of reporters indicative for DNA damage. At cytoxic concentrations incerased induction was seen in reporters that are indicative of oxicdative strress and unfolded protein.

 

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

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

Justification for type of information:

See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

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 7

Endpoint:

genetic toxicity in vitro, other

Remarks:

ToxTracker Assay

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2016-09-19 until 2016-10-06

Reliability:

2 (reliable with restrictions)

Justification for type of information:

See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

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

No data is available on the product Sodium diisobutylnaphthalenesulphonate (ANS DIB; C8-alkyl naphthalene sulfonate) itself. Evaluation is based on read-across to the comparable C7-alkyl naphthalene sulfonate (ANS IP) and other substances in the Alkyl Naphthalene Sulfonates (ANS) category. See chapter 13 for support for read-across within the category of Alkyl Naphthalene Sulfonates (ANS).

 

Ames:

Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt (ANS IP; C7-alkyl naphthalene sulfonate) 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 sulfonateis non-mutagenic in the bacterial reverse mutation assay.

 

ToxTracker:

C7-alkyl naphthalene sulfonate 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 sulfonate 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 sulfonateis provided by the available data on the comparable ANS N (‘high nonene’). Testing on this ANS N confirmed 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.

 

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 sulfonate and both ANS N (‘high nonene’) and ANS N (‘Low nonene’): 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.

Justification for classification or non-classification

C7-alkyl naphthalene sulfonatewas 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 sulfonates indicate that genotoxic properties are rather unlikely.

Lack of genotoxicity is further confirmed with cross-reading fromANS N, 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.