Naphthalene sulfonic acid, reaction products with isobutanol,sodium salts

Endpoint summary

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

Reference

Reference 1

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.

Reference 2

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 Prediction²
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%.

Reference 3

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.

Reference 4

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 (I_max) induction of 1.26 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 100.9%. Therefore, no EC_1.5 could be calculated.

In the second experiment, no luciferase activity induction above 1.5 was observed. A max luciferase activity (I_max) induction of 1.40 was determined at the test item concentrations of 250.00 µM. The corresponding cell viabilities were 59.8%. Therefore, no EC_1.5 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 C_10-13-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.