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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(2003)
Deviations:
no
Qualifier:
according to
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. certificate)
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.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Sodium alkylnaphthalene sulfonate
- Chemical name: Aromatic hydrocarbons, C10-13, reaction products with branched nonene, sulphonated sodium salts
- CAS Number: 1258274-08-6
- Description: Slightly yellow powder (determined at NOTOX)
- Lot/batch No.: 1238887
- Expiration date of the lot/batch: 03 May 2021
- Storage condition of test material: At room temperature in the dark under nitrogen
- Test substance handling: Flush container with nitrogen after handling
- pH: 7.5 – 10 at concentration of 5%

In vivo test system

Test animals

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

Study design: in vivo (non-LLNA)

Inductionopen allclose all
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%
Challengeopen allclose all
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.

Results and discussion

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.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
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 group
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 group
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 group
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..

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.