Registration Dossier

Administrative data

Description of key information

No data is available on the product Sodium diisobutylnaphthalenesulphonate (ANS DIB; C8-alkyl naphthalene sulfonate) itself. 

C7-alkyl naphthalene sulfonate (ANS IP) was tested in three in vitro studies: The DPRA was not possible to evaluate due to co-elution); KeratinoSens was negative and h-CLAT was also negative. Based on the two-out-of-three approach it was concluded that the substance is not sensitising to skin.

A Buehler test performed according to OECD 406 guideline and GLP guidelines ANS N (‘high nonene’) also showed no sensitising potential.

Structure profile further supports the lack of sensitising properties, and none of the relevant profilers forpeptide depletion and protein bindingin QSAR Toolbox (v 4.2) triggered a concern. QSARs provide mixed results for skin sensitisation, but it is clearthat there is a lack of comparable structures showing sensitisation for statistical QSAR models, and in that respect the DEREK expert evaluation, predicting non-sensitizing, bears the most weight.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
March 06, 2012 - May 25, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD/EC guideline and 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 406 (Skin Sensitisation)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(2003)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions.
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
An old study in which the sensitization of a preparation ofalkylnaphthalene sulfonate (ANS) sodium salt in LAS (C10-16 alkyl benzene sulphonic acid, sodium salt) was evaluated according to the Buehler method, resulted to difficult interpretable results. Already during induction phase, maximum responses were obtained at the second and third topical exposures. Especially since there are no reports on sensitisation among workers handling these products since the 1950´s this casts great doubts on the results of this study. Following the advice of a consulted expert (David Basketter) a new study on the “pure” ANS material was performed. “REACH might require that a LLNA is the first choice assay, but in this case a powerful argument could be made for the conduct of a Buehler test, since the need is to understand the earlier positive Buehler result and changing more than one variable (ie test substance, test vehicle and test protocol) is very bad science.”. Additionally, irritating surfactants have been shown to result to false positive results in LLNA.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany.
- Age at study initiation: Young adult animals (approx. 13 weeks old at first induction)
- Weight at study initiation: 584 - 758 g
- Housing: Group housing of maximally 5 animals per labeled cage containing sterilized sawdust as bedding material and shelters as cage enrichment.
- Diet: Free access to Complete breeding diet for guinea pigs (SSNIFF® MS-Z, V2273; SSNIFF® Spezialdiäten GmbH, Soest, Germany). Hay was provided at least twice a week.
- Water: Free access to tap water.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 - 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Preliminary irritation study: Induction 1, 2, 5, 10, 30 and 70%, and challenge 5%

Main study: Induction 5%, and challenge 2 and 5%
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Preliminary irritation study: Induction 1, 2, 5, 10, 30 and 70%, and challenge 5%

Main study: Induction 5%, and challenge 2 and 5%
No. of animals per dose:
Preliminary irritation study: 5 (total)
Main study: Test substance group 20 animals and control group 10 animals
Details on study design:
RANGE FINDING TESTS: Initially, a series of four test substance concentrations was used (5, 10, 30 and 70%), the highest concentration being the maximum concentration that could technically be applied. The resulting dermal reactions were assessed for irritation 24 and 48 hours after exposure. Animals treated at 30, 10 and 5% again received a dermal application on Day 8, using the same procedures as used for Day 1 to assess occurrence of any delayed irritation. A 70% concentration was not administered since this resulted in sign of necrosis after application on Day 1.
To address whether sensitization could occur during the induction phase, animals also received a simultaneous epidermal application of a 5% test substance concentration (0.1 mL each) to the clipped, contralateral flank of all animals, using Patch Test Plasters (Curatest®) on Day 14. A 5% test substance concentration was non-irritating after exposure on Day 1. Based on the results, one animal was treated with two lower concentrations (2 and 1%) on Days 1, 8 and 15, using the same procedures as followed for animals exposed at the highest concentrations. A macroscopic examination was conducted for all animals used in the preliminary irritation Study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 hours, the skin was cleaned of residual test substance using tap water. The treated area was assessed for irritation at 24 and 48 hours after removal of the bandage. This procedure was repeated on Days 8 and 15.
- Test group: 20 females
- Control group: 10 females
- Site: The left side of the scapular region was clipped (2x3 cm).
- Concentrations: 0.5 mL of a 5% test substance concentration

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: two weeks after the final induction
- Exposure period: 6 hours, the skin was cleaned of residual test substance and vehicle using water.
- Test group: 20 females
- Control group: 10 females
- Site: The left and right flank of all animals was clipped.
- Concentrations: The left flank was epidermally treated with a 5% test substance concentration, and the right flank was epidermally treated with a 2% test substance concentration and the vehicle (0.1 mL each).
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressings.

OTHER:
Observations:
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body weights: Prior to start and at termination of the study.
- Irritation: irritation was assessed according to the numerical scoring system according to OECD 406.
Positive control substance(s):
yes
Remarks:
A reliability check was carried out (performed in November/December 2011) with Alpha-Hexylcinnamaldehyde.
Positive control results:
The skin reactions observed in five experimental animals in response to the 50% alpha-hexylcinnamidcaldehyde concentration in the challenge phase were considered indicative of sensitization, based on the absence of any response in the control animals.The results of the reliability check lead to a sensitization rate of 50 per cent indicating the animal model used is an appropriate model to evaluate the sensitising potential.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
2%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 2%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study..
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
2%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study..

Preliminary irritation study:

Treatment with a 10, 30 and 70% test substance concentration resulted in necrosis of the treated skin area. Treatment with a 5% test substance concentration resulted in superficial necrosis around the hair follicles of the treated skin area in one of the two treated animals, at 48 hours after the second exposure. The other animal at this concentration showed scaliness of the treated skin area after the second exposure. Treatment with a 1% and 2% test substance concentration did not result in signs of irritation. Based on these results, a 5% test substance concentration was selected for the three epidermal induction exposures.

The 2% test substance concentration was the highest non-irritating concentration during 3 subsequent dermal exposures, and was therefore selected for the challenge phase, next to a 5% test substance concentration that was non-irritating after a single exposure. Comparison of the skin reactions to exposure of a 5% test substance concentration applied to control animals provided additional information on the skin sensitizing potential of the test substance.

A challenge expose with a 5% test substance concentration on Day 14 yielded no response in the animals previously exposed to a 5%, 10%, 30% and/or 70% test substance concentration on Days 1 and/or 8.

Main study:

- Induction phase: No irritation was noted in any control animal during the induction phase. After the first exposure (Day 1), no signs of irritation were noted in any experimental animal treated with a 5% test substance concentration, which was in line with the preliminary irritation study results. After the second exposure (Day 8), 6 out of 20 animals showed an irritation response consisting of scaliness and/or slight erythema, which was reversible within 48 hours after exposure in all affected animals. After the last exposure (Day 15), 14 out of 20 experimental animals showed an irritation response varying from scaliness to well-defined erythema, and one animal showed necrosis. These irritation responses after the last exposure were generally not reversible within 48 hours after exposure.

- Challenge phase: One animal showed a grade 1 skin reaction after challenge with a 2% and 5% concentration at 24 and/or 48 hours after removal of the bandage. No skin reactions were evident after the challenge exposure in the other experimental animals and all control animals. The only animal showing a positive response after challenge with a 2% and 5% concentration, also showed the most pronounced response during the induction phase.

- Toxicity/mortality: No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

- Body weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this Buehler test performed according to OECD 406 guideline and GLP guidelines Sodium alkylnaphthalene sulfonate was found to be non-sensitisng.
Executive summary:

An old study in which the sensitization of a preparation ofalkylnaphthalene sulfonate (ANS) sodium salt in LAS (C10-16 alkyl benzene sulphonic acid, sodium salt) was evaluated according to the Buehler method, resulted to difficult interpretable results. Already during induction phase, maximum responses were obtained at the second and third topical exposures. Especially since there are no reports on sensitisation among workers handling these products since the 1950´s this casts great doubts on the results of this study. Following the advice of a consulted expert (David Basketter) a new study on the “pure” ANS material was performed. “REACH might require that a LLNA is the first choice assay, but in this case a powerful argument could be made for the conduct of a Buehler test, since the need is to understand the earlier positive Buehler result and changing more than one variable (ie test substance, test vehicle and test protocol) is very bad science.”

Assessment of Contact Hypersensitivity to Sodium alkylnaphthalene sulfonate in the Albino Guinea Pig (Buehler Test).

The study was carried out based on the guidelines and test method described in:

-      OECD No. 406 (1992), "Skin Sensitization"

-      EC No 440/2008; B6: "Skin Sensitization: Buehler Test”.

-      EPA OPPTS 870.2600 (2003) “Skin Sensitization”

-      JMAFF: Japanese Test Guidelines (2000) including the most recent partial revisions.

The Buehler type of sensitization test was selected at request of the sponsor.

Test substance concentrations selected for the Main study were based on the results of a preliminary study. In the Main study, twenty experimental animals were epidermally treated on three occasions (Days 1, 8 and 15) with a 5% test substance concentration and ten control animals were similarly treated, but with vehicle alone (water). Two weeks after the last induction exposure, all animals were challenged with a 2% and 5% test substance concentration and the vehicle.

 

During the induction phase, no irritation was noted in any control animal. After the first exposure (Day 1), no signs of irritation were noted in any experimental animal treated with a 5% test substance concentration, which was in line with the preliminary irritation study results. After the second exposure (Day 8), 6 out of 20 animals showed an irritation response consisting of scaliness and/or slight erythema, which was reversible within 48 hours after exposure in all affected animals. After the last exposure (Day 15), 14 out of 20 experimental animals showed an irritation response varying from scaliness to well-defined erythema, and one animal showed necrosis. These irritation responses after the last exposure were generally not reversible within 48 hours after exposure.

In the challenge phase, one animal showed a grade 1 skin reaction after exposure with a 2% and 5% concentration at 24 and/or 48 hours after removal of the bandage. No skin reactions were evident after the challenge exposure in the other experimental animals and all control animals. The only animal showing a positive response after challenge with a 2% and 5% concentration, also showed the most pronounced response during the induction phase.

 

The skin reactions observed in response to the 2% and 5% test substance concentration in one out of twenty experimental animals in the challenge phase were considered indicative of sensitization, based on the absence of any response in the control animals. These results indicate a sensitization rate of 5%. It should be noted that the only animal showing a positive response after challenge with a 2% and 5% concentration, also showed the most pronounced response (necrosis) during the induction phase. In the preliminary irritation study, however, a challenge exposure on Day 14 with a 5% test substance concentration yielded no response in the animals previously exposed to a 5%, 10%, 30% and/or 70% test substance concentration on Days 1 and/or 8. This suggests that the notable irritation responses seen among these animals had not provoked a sensitization response.

 

Based on these results Sodium alkylnaphthalene sulfonate does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the:

- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011),

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Endpoint:
skin sensitisation: in chemico
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
2017-11-09 to 2017-12-09
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 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
other: (in chemico) reactivity against synthetic peptides with a thiol or amino group
Specific details on test material used for the study:
Name: Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt
Batch No.: 1452486
Chemical name: Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt
CAS No: 68909-82-0
Purity (Actives): 75.98%
Further Components: Salts (alcohols insoluble): 15.61%
Unsulfonated oil (ether ext.): 7.42%
Moisture: 0.99%
Physical State: solid
Average Molecular Weight: 346 g/mol
Colour: tan
Storage Conditions: room temperature
Stability: stable
Expiry Date: 07 June 2021
Safety Precautions: The routine hygienic procedures are sufficient to assure personnel health and safety.
Details on the study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.07%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Run / experiment:
other: lysine run
Parameter:
other: mean peptide depletion [%]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

4862.8555

0.5340

4355.7368

0.5340

STD2

2384.9666

0.2670

2207.4150

0.2670

STD3

1183.9525

0.1335

1077.4733

0.1335

STD4

582.0698

0.0667

534.9317

0.0667

STD5

301.6335

0.0334

267.5728

0.0334

STD6

159.0007

0.0167

133.3614

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1453.8585

0.1612

68.18

67.81

0.64

0.94

1504.0075

0.1667

67.08

1453.6510

0.1612

68.18

Test Item*

--

--

--

--

--

--

--

--

--

--

--

--

Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1660.4912

0.2034

59.71

60.33

0.55

0.92

1627.6788

0.1994

60.51

1616.5613

0.1980

60.78

Test Item

--

--

--

--

--

--

--

--

--

--

--

--

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model 1

Mean Cysteine andLysine PPD

Reactivity Class

DPRA Prediction²

0.00% PPD 6.38%

 No or Minimal Reactivity

Negative

6.38% < PPD 22.62%

Low Reactivity

Positive

22.62% < PPD 42.47%

Moderate Reactivity

42.47% < PPD 100%

High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD

ReactivityClass

DPRA Predictio

0.00% PPD 13.89%

No or Minimal Reactivity

Negative

13.89% < PPD 23.09%

Low Reactivity

Positive

23.09% < PPD 98.24%

Moderate Reactivity

98.24% < PPD 100%

High Reactivity

Categorization of the Test Item

Prediction Model

Prediction Model 1
(Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)

Prediction Model 2
(Cysteine Peptide / Test Item Ratio: 1:10)

Test Substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

 

 

 

 

 

 

Positive Control

64.07

High Reactivity

sensitiser

67.81

Moderate Reactivity

sensitiser

Interpretation of results:
other: Study is part of a weight of evidence approach and is not used for classification on its own.
Remarks:
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Conclusions:
In this study under the given conditions the test item could not be classified due to co-elution of the test item with the both peptide peaks.
Executive summary:

In the present study Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt was dissolved in dist. water, based on the results of the pre-experiments.

Based on the average molecular weight of 346 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Turbidity was observed for the samples of the test item (including the co-elution control of the test item). Precipitation was observed for the samples of the positive control (including the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations and turbidity were regarded as insignificant.

As co-elution of the test item with both peptide peaks was observed and evaluation of the peptide depletion was not possible. Therefore, the result must be considered as inconclusive

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.07%.

Endpoint:
skin sensitisation: in vitro
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
2016-11-21 to 2017-07-06
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:
other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016
Qualifier:
according to guideline
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
Details on the study design:
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.
Positive control results:
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all of the three experiments.
The threshold of 150% for CD86 (464% experiment 1; 556% experiment 2, 196% experiment 3) and
200% for CD54 (947% experiment 1; 703% experiment 2, 241% experiment 3) were clearly exceeded.
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
153
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
184
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
117
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
176
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
97
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
80
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

Table3:  Results of the Dose Finding Assay

Sample

Experiment 1

Experiment 2

Concentration applied [µg/ml]

Cell Viability [%]

Concentration applied [µg/ml]

Cell Viability [%]

Medium Control

0.00

98.00

0.00

97.50

Solvent Control

-

98.00

-

97.50

Naphthalenesulfonic acid, bis(1-methylethyl)-,methyl derivs., sodium salt

7.81

98.00

7.81

97.30

15.63

97.60

15.63

96.90

31.25

97.90

31.25

97.20

62.50

97.80

62.50

96.80

125.00

68.80

125.00

67.60

250.00

2.90

250.00

6.60

500.00

0.10

500.00

0.10

1000.00

0.20

1000.00

0.00

Calculated CV75 [µg/mL]

107.78

104.86

Mean CV75 [µg/mL]

106.32

SD CV 75 [µg/mL]

2.07

Table4:  CD54 and CD86 Expression Experiment 1

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

98.1

98.4

98.0

1424

939

796

628

143

100

100

179

118

DMSO Control

0.20%

97.8

98.2

97.8

1621

960

790

831

170

132

119

205

122

DNCB

4.00

86.8

88.0

87.2

4745

2503

893

3852

1610

464

947

531

280

Naphthalenesulfonic acid, bis(1-methylethyl)-,

127.59

71.3

69.0

72.7

2743

2054

1909

834

145

133

101

144

108

106.33

92.7

92.8

91.3

2440

1745

1482

958

263

153

184

165

118

88.60

97.0

96.8

97.1

2269

1579

1387

882

192

140

134

164

114

73.84

97.3

96.9

96.8

2075

1524

1368

707

156

113

109

152

111

61.53

97.9

97.6

97.9

1682

1289

1159

523

130

83

91

145

111

51.28

97.9

98.0

97.9

1462

912

761

701

151

112

106

192

120

42.73

97.8

98.2

98.1

1937

1227

1122

815

105

130

73

173

109

35.61

98.3

98.3

97.9

1884

1237

1045

839

192

134

134

180

118

 

Table5: CD54 and CD86 Expression Experiment 2

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

IgG Isotype

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

96.9

97.3

96.7

1476

1025

864

612

161

100

100

171

119

DMSO Control

0.20%

96.5

96.4

96.4

1562

1033

858

704

175

115

109

182

120

DNCB

4.0

84.3

85.4

85.4

4737

2057

826

3911

1231

556

703

573

249

Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt

127.59

72.5

70.8

71.3

2469

2000

1840

629

160

103

99

134

109

106.33

91.8

91.0

90.5

2300

1860

1583

717

277

117

172

145

117

88.60

94.0

93.9

94.1

2057

1675

1392

665

283

109

176

148

120

73.84

96.1

95.8

95.8

1920

1563

1312

608

251

99

156

146

119

61.53

96.5

95.9

95.8

1778

1487

1328

450

159

74

99

134

112

51.28

96.7

96.4

96.0

1737

1353

1203

534

150

87

93

144

112

42.73

96.6

96.5

96.5

1668

1294

1156

512

138

84

86

144

112

35.61

96.5

96.6

96.7

1731

1257

1078

653

179

107

111

161

117

 

Table6: CD54 and CD86 Expression Experiment 3

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

IgG Isotype

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

96.5

96.7

96.7

1656

1022

477

1179

545

100

100

347

214

DMSO Control

0.20%

96.7

96.6

96.6

1692

982

444

1248

538

106

99

381

221

DNCB

4.0

78.0

79.9

79.4

2966

1823

526

2440

1297

196

241

564

347

Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt

127.6

64.9

63.7

61.1

2419

1646

1276

1143

370

97

68

190

129

106.33

94.5

94.4

94.6

2010

1465

1051

959

414

81

76

191

139

88.60

95.8

96.3

96.4

1896

1404

968

928

436

79

80

196

145

73.84

96.6

96.7

96.8

1801

1223

858

943

365

80

67

210

143

61.53

96.9

96.8

96.7

1595

1235

841

754

394

64

72

190

147

51.28

96.7

94.6

96.9

1693

1144

763

930

381

79

70

222

150

42.73

97.4

97.4

96.8

1582

1093

680

902

413

77

76

233

161

35.61

96.9

97.2

97.3

1583

1086

655

928

431

79

79

242

166

 

Table7:  Acceptance Criteria

Acceptance Criterion

range

Experiment 1

pass/fail

Experiment 2

pass/fail

Experiment 3

pass/fail

cell viability solvent controls [%]

>90

97.8 – 98.4

pass

96.4 – 97.3

pass

96.5 – 96.7

pass

number of test dosed with viability >50% CD86

≥4

8

pass

8

pass

8

pass

number of test dosed with viability >50% CD54

≥4

8

pass

8

pass

8

pass

number of test dosed with viability >50% IgG1

≥4

8

pass

8

pass

8

pass

RFI of positive control of CD86

≥150

464

pass

556

pass

196

pass

RFI of positive control of CD54

≥200

947

pass

703

pass

241

pass

RFI of solvent control of CD86

<150

132

pass

115

pass

106

pass

RFI of solvent control of CD54

<200

119

pass

109

pass

99

pass

MFI ratio IgG1/CD86 for medium control [%]

>105

179

pass

171

pass

347

pass

MFI ratio IgG1/CD86 for DMSO control [%]

>105

205

pass

182

pass

381

pass

MFI ratio IgG1/CD54 for medium control [%]

>105

118

pass

119

pass

214

pass

MFI ratio IgG1/CD54 for DMSO control [%]

>105

122

pass

120

pass

221

pass

 

Interpretation of results:
other: Study is part of a weight of evidence approach and is not used for classification on its own.
Remarks:
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in two out of three independent experiment runs.Therefore the test item is considered to be a non-sensitiser.
Executive summary:

In the present study Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt was dissolved in 0.9% NaCl. A CV75 of 106.32 ± 2.07 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps:

127.59, 106.33, 88.60, 73.84, 61.53, 51.28, 42.73, 35.61 µg/mL

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Slight cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 71.3% (CD86), 69.0% (CD54) and 72.7% (isotype IgG1 control) in the first experiment, 72.5% (CD86), 70.8% (CD54) and 71.3% (isotype IgG1 control) in the second experiment and to 64.9% (CD86), 63.7% (CD54) and 61.1% (isotype IgG1 control) in the third experiment.

The expression of the cell surface marker CD86 was upregulated to 153% in the first experiment in one concentration. The upregulation above the threshold of 150% was observed at a concentration of 106.33 µg/mL. In experiments 2 and 3 no upregulation of CD 86 above the threshold was observed. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all of the three experiments. The threshold of 150% for CD86 (464% experiment 1; 556% experiment 2, 196% experiment 3) and 200% for CD54 (947% experiment 1; 703% experiment 2, 241% experiment 3) were clearly exceeded.

Endpoint:
skin sensitisation: in vitro
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
2016-11-28 to 2017-12-05
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 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Details on the study design:
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by
addressing the second molecular key event of the adverse outcome pathway (AOP), namely
activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line
KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared
to the respective solvent controls is used to support discrimination between skin sensitisers
and non-sensitisers.

Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.82 (experiment 1), 2.38 (experiment 2).
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
1.26
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: cell viability [%]
Value:
100.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
n the first experiment no luciferase activity induction above 1.5 was observed. A max luciferase activity (Imax) induction of 1.26 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 100.9%. Therefore, no EC1.5 could be calculated.
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
1.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
59.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
In the second experiment, no luciferase activity induction above 1.5 was observed. A max luciferase activity (Imax) induction of 1.40 was determined at the test item concentrations of 250.00 µM. The corresponding cell viabilities were 59.8%. Therefore, no EC1.5 could be calculated.
Other effects / acceptance of results:
Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition which consisting of 6 wells.

The controls fullfilled the validity criteria of the test.

Table1: Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell Viability [%]

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

100

100

100

0.0

Positive Control

4.00

106.5

97.6

102.1

6.2

8.00

120.0

94.6

107.3

18.0

16.00

127.0

113.1

120.0

9.8

32.00

125.9

112.4

119.1

9.5

64.00

147.7

120.5

134.1

19.2

Test Item

0.98

77.2

108.3

92.7

22.0

1.95

102.5

78.2

90.3

17.1

3.91

103.8

77.0

90.4

18.9

7.81

100.9

79.7

90.3

15.0

15.63

109.4

68.6

89.0

28.9

31.25

108.6

73.7

91.1

24.6

62.50

115.4

80.5

98.0

24.7

125.00

173.8

125.2

149.5

34.3

250.00

90.6

59.8

75.2

21.7

500.00

-0.3

0.1

-0.1

0.3

1000.00

-0.5

0.3

-0.1

0.6

2000.00

-0.3

0.4

0.0

0.5

Table2: Induction of Luciferase Activity Experiment 1

Experiment 1

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

Positive Control

4.00

1.15

1.02

1.15

1.11

0.08

8.00

1.16

1.31

1.28

1.25

0.08

16.00

1.57

1.40

1.52

1.50

0.09

32.00

1.66

1.57

1.44

1.55

0.11

*

64.00

2.96

3.00

2.49

2.82

0.29

*

Test Item

0.98

1.50

1.04

0.97

1.17

0.29

1.95

1.02

0.87

1.09

1.00

0.11

3.91

1.10

1.19

1.00

1.10

0.09

7.81

1.19

0.99

1.60

1.26

0.31

15.63

1.00

1.12

1.02

1.05

0.07

31.25

1.02

0.79

1.00

0.94

0.13

62.50

0.76

0.76

0.83

0.78

0.04

125.00

0.52

0.67

0.63

0.61

0.08

250.00

0.80

1.60

1.28

1.23

0.40

500.00

0.01

0.02

0.06

0.03

0.02

1000.00

0.00

0.00

0.00

0.00

0.00

2000.00

0.00

0.00

0.00

0.00

0.00

* = significant induction according to Student’s T-test, p<0.05

Table3: Induction of Luciferase Activity Experiment 2

Experiment 2

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

Positive Control

4.00

0.94

0.94

1.27

1.05

0.19

8.00

1.43

1.09

1.21

1.24

0.17

16.00

1.28

1.28

1.54

1.36

0.15

32.00

1.65

1.36

1.81

1.60

0.23

*

64.00

2.20

2.03

2.91

2.38

0.47

*

Test Item

0.98

0.90

0.85

1.19

0.98

0.18

1.95

0.91

0.85

0.98

0.92

0.07

3.91

0.70

0.82

1.10

0.88

0.20

7.81

0.84

0.79

1.07

0.90

0.15

15.63

0.74

0.95

1.15

0.95

0.21

31.25

0.91

0.78

1.08

0.92

0.15

62.50

0.74

0.74

1.07

0.85

0.19

125.00

0.62

0.59

0.86

0.69

0.15

250.00

1.18

1.22

1.80

1.40

0.34

500.00

0.11

0.04

0.06

0.07

0.04

1000.00

0.00

0.00

0.00

0.00

0.00

2000.00

0.00

0.00

0.00

0.00

0.00

* = significant induction according to Student’s T-test, p<0.05

Table4: Induction of Luciferase Activity – Overall Induction

 

Concentration [µM]

Fold Induction

Significance

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

Positive Control

4.00

1.11

1.05

1.08

0.04

8.00

1.25

1.24

1.25

0.01

16.00

1.50

1.36

1.43

0.09

32.00

1.55

1.60

1.58

0.04

*

64.00

2.82

2.38

2.60

0.31

*

Test Item

0.98

1.17

0.98

1.08

0.13

1.95

1.00

0.92

0.96

0.06

3.91

1.10

0.88

0.99

0.15

7.81

1.26

0.90

1.08

0.25

15.63

1.05

0.95

1.00

0.07

31.25

0.94

0.92

0.93

0.01

62.50

0.78

0.85

0.82

0.05

125.00

0.61

0.69

0.65

0.06

250.00

1.23

1.40

1.32

0.12

500.00

0.03

0.07

0.05

0.03

1000.00

0.00

0.00

0.00

0.00

2000.00

0.00

0.00

0.00

0.00

* = significant induction according to Student’s T-test, p<0.05

Table5: Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

n.d.

n.d.

n.d.

n.d.

Imax

1.26

1.40

1.33

0.10

IC30[µM]

306.66

125.80

216.23

127.88

IC50[µM]

361.70

291.16

326.43

49.88

n.d. = not determined

Interpretation of results:
other: Study is part of a weight of evidence approach and is not used for classification on its own.
Remarks:
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
Executive summary:

In the present study Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt was dissolved in distilled water. Since the test item had no defined molecular weight, the test was to be performed using a pro forma molecular weight of 200 g/mol. Based on this, a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2.

These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. The following concentration range was tested in the assay:

2000.00, 1000.00, 500.00, 250.00, 125.00, 61.50, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment no luciferase activity induction above 1.5 was observed. A max luciferase activity (Imax) induction of 1.26 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 100.9%. Therefore, no EC1.5 could be calculated.

In the second experiment, no luciferase activity induction above 1.5 was observed. A max luciferase activity (Imax) induction of 1.40 was determined at the test item concentrations of 250.00 µM. The corresponding cell viabilities were 59.8%. Therefore, no EC1.5 could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

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

 

DPRA:

The reactivity of Naphthalenesulfonic acid, bis(1-methylethyl)-, methyl derivs., sodium salt (C7-alkyl naphthalene sulfonate; ANS IP) towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was evaluated in the Direct Peptide Reactivity Assay (DPRA) in a GLP compliant study according to the most recent OECD 442C guideline. After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and diode array detection (DAD) detection at 220 nm and 258 nm. All validation parameters were within the acceptability criteria for the DPRA.

 

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24h incubation period but prior to the HPLC analysis turbidity was observed for the samples of the test item (including the co-elution control of the test item). Precipitation was observed for the samples of the positive control (including the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24h incubation period but prior to the HPLC analysis no precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

As co-elution of the test item with both peptide peaks was observed and evaluation of the peptide depletion was not possible.

 

KeratinoSens:

C7-alkyl naphthalene sulfonate was tested for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay in aGLP compliantstudy according to the most recent OECD 442D guideline. Two independent experiments were performed which both passed the acceptance criteria for the positive control Cinnamic aldehyde and showing a dose response relation, and the average coefficient of for the negative control (DMSO) was below 20%.

Cytotoxic effects were seen from 250 µM, showing a cell viability of 75.2%. Higher concentrations were too cytotoxic for evaluation.

In the first experiment no luciferase activity induction above 1.5 was observed. A max luciferase activity (Imax) induction of 1.26 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 100.9%. Therefore, no EC1.5 could be calculated.

In the second experiment, no luciferase activity induction above 1.5 was observed. A max luciferase activity (Imax) induction of 1.40 was determined at the test item concentrations of 250 µM. The corresponding cell viabilities were 59.8%. Therefore, no EC1.5 could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs.

 

h-CLAT:

C7-alkyl naphthalene sulfonate wasevaluated forthe ability to increase the expression levels of CD54 and CD86 cell surface marker in THP-1 cells in human Cell Line Activation Testassay, performedin a GLP compliant study according to the most recent OECD 442Eguideline.

Three independent experiments were performed.All experimentspassed the acceptance criteria and inall experimentsthe positive and negative control were considered valid.

Cytotoxicity was evidenced in a dose finding assay by decreasing cell viability after test concentration of 62.50 µg/mL, with 68.8% and 67,2% viability at 125 µg/mL in two separate experiments.Higher concentrations were too cytotoxic for evaluation. The main experiment was performed covering a concentration range from 127.59 µg/mL to 35.61 µg/mL.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel.

Slight cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 61.1% to 72.7%.

The expression of the cell surface marker CD86 was upregulated above the threshold of 150% (to 153%) in the first experiment in one concentration (of 106.33 µg/mL). In experiments 2 and 3 no upregulation of CD 86 above the threshold was observed. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all of the three experiments.

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in two out of three independent experiment runs. Therefore the test item is considered to be a non-sensitizer.

 

The results of these in vitro studies indicate a lack of sensitizing potential for C7-alkyl naphthalene sulfonate. Also available data onthe very similar C10-13-alkylnaphthalene sulfonate (ANS N (‘high nonene’)) point to a lack of skin sensitisation potential when tested ina Buehler test performed according to OECD 406 guideline and GLP guidelines.

 

Structure profile further supports the lack of sensitising properties, and none of the relevant profilers forpeptide depletion and protein bindingin QSAR Toolbox (v 4.2) triggered a concern. QSARs provide mixed results for skin sensitisation.

- Derek Nexus v.6.0.1: non-sensitizer, no misclassified or unclassified Features;

- VEGA Skin Sensitization model (CAESAR version 2.1.6): NON-Sensitizer (low reliability- compound is outside the Applicability Domain)

- BIOVIA Discovery Studio ADME & TopKat Toxicity Package: Strong sensitizer, but no structurally similar compounds are indicated.

It is clear that there is a lack of comparable structures showing sensitisation for statistical QSAR models, and in that respect the DEREK expert evaluation bears the most weight.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

No study is available. At present, recognised and validated animal models for the testing of respiratory hypersensitivity are not available. Exposure of humans via inhalation is not likely taking into account the vapour pressure of the substance (1.7 × 10-4 Pa at 25 °C) and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.  

As chemical respiratory sensitisers also elicit positive results in predictive tests for contact sensitisation, a negative outcome for dermal sensitisation is also predictive for non-respiratory sensitisation of the substance.

Justification for classification or non-classification

Three in vitro studies (DPRA, KeratinoSens and h-CLAT) showed no concerns for sensitisation.

 

In a Buehler test performed according to OECD 406 guideline and GLP guidelines Sodium C10-13-alkylnaphthalene sulfonate (ANS N (‘high nonene’)) was found to be non-sensitising. Structure profile further supports the lack of sensitising properties.

 

As chemical respiratory sensitizers also elicit positive results in predictive tests for contact sensitisation, a negative outcome for dermal sensitisation is also predictive for non-respiratory sensitisation of the substance.