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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 19, 2011 to October 27, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Refer to the section 13 of IUCLID dataset for details on the read across justification. The skin sensitisation study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test substance was considered to have surface active properties and skin irritants with surface active properties tend to produce false positive results in LLNA study. Hence it was decided to conduct a non-LLNA study (GPMT) to evaluate the skin sensitisation potential.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles river (F-69592 L'Arbresle)
- Age at study initiation: 4 weeks old
- Weight at study initiation: 255- 292 g
- Housing: the animals were housed either in groups of 2 or individually in polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted a stainless steel lid with a feeding device and drinking device of 500 mL.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: a minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19- 25°C
- Humidity (%): 30- 70%
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

PREPARATION OF ANIMALS
The animals were carefully shorn before each test substance application:- On the inter-scapular zone for the induction phase,- On the dorso-lumbar zone for the challenge phase.At least 3 hours before the first reading (challenge phase) they were shorn a second time in this dorso-lumbar zone.The animals were weighed at the beginning and at the end of the study.
Route:
intradermal and epicutaneous
Vehicle:
other: The test substance was used freshly prepared in isotonic sodium chloride for the intradermic injections and in distilled water for the topical applications.
Concentration / amount:
Main study: Induction: Intradermic injection at 0.1% in isotonic soidum chloride and topical application at 50% in distilled water.Challenge: 0.5% and 0.25% in distilled water
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: the test item was used freshly prepared in isotonic sodium chloride for the intradermic injections and in distilled water for the topical applications.
No.:
#2
Concentration / amount:
Main study: Induction: Intradermic injection at 0.1% in isotonic soidum chloride and topical application at 50% in distilled water.Challenge: 0.5% and 0.25% in distilled water
No. of animals per dose:
Group 1 (negative control): 5 Group 2 (treated): 10
Details on study design:
RANGE FINDING TESTS:

Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC):

This test was conducted for the purpose of defining a MNNC of the test substance which, on intradermal injection during the induction phase, does not risk causing too great a lesion (non-necrotizing concentration), should be well tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. One animal received on both sides of the spine, a volume of 0.1 mL of the test substance, at 4 concentrations: diluted at 25, 10, 5 and 2.5% in isotonic sodium chloride in order to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after injection. Due to the necrosis registered, a new animal received on both sides of the spine, a volume of 0.1 mL of the test substance, at 4 concentrations: diluted at 1, 0.5, 0.1 and 0.05% in isotonic sodium chloride in order to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after injection. The results were confirmed with a new animal. One animal received on both sides of the spine, a volume of 0.1 mL of the test substance, at 2 concentrations: diluted at 0.1 and 0.05% in isotonic sodium chloride in order to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after injection. Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC): This test, which allowed to evaluate the irritant potential of the test substance, defined whether an application of sodium lauryl sulphate would be needed during topical induction phase. The test substance was applied on the dorso-lumbar zone of 1 animal shorn beforehand, with occlusive dressing for 24 h, at 4 different concentrations: diluted at 50% in distilled water and 25, 10 and 5% in isotonic sodium chloride. The results were confirmed with a new animal. The animal received the test substance in the same experimental conditions, at 4 different concentrations: diluted at 50, 25, 10 and 5% in distilled water. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after removal of the dressing. Determination by topical application of the Maximal Non Irritant Concentration (MNIC): This test was carried out for the purpose of determining the MNIC of the test substance without risk of an irritant effect during the challenge phase. Three animals were treated identically to the animals from Group 1 (negative control) for the induction phase (i.e. isotonic sodium chloride and distilled water). During the challenge phase, the animals were treated with the test substance placed onto the selected treatment sites at concentrations: 4, 2, 1 and 0.5% in distilled water and covered with an occlusive dressing for a period for 24 h. A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 h after removal of the occlusive dressing.

Main study:
Group 1 (negative control): 5 male guinea pigs
Group 2 (treated): 10 male guinea pigs

Induction phase: 1st Intradermal Induction: Day 0: after shearing the scapular zone, 3 pairs of intradermal injections (ID) of 0.1 mL were made on the scapular zone such that an injection from each pair was placed either side of the spine as follows:
Group 1 (negative control): 2 ID: Freund’s Complete Adjuvant diluted at 50% in isotonic sodium chloride; 2 ID: isotonic sodium chloride; 2 ID: a mixture with equal volumes v/v of Freund’s Complete Adjuvant at 50% and isotonic sodium chloride.
Group 2 (treated): 2 ID: Freund’s Complete Adjuvant diluted at 50% in isotonic sodium chloride; 2 ID: test substance at 0.1% in isotonic sodium chloride; 2 ID: a mixture with equal volumes v/v of Freund’s Complete Adjuvant at 50% and the test substance at 0.2% in isotonic sodium chloride.
Day 1: Skin reaction evaluation 2nd topical induction:
Day 6: the scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulphate at 10% in thick vaseline, in order to create a local irritation.
Day 7: a topical application under occlusive dressing for 48 h was performed on the injection sites of each animal. A control of the local irritation was done just before the topical application.
Group 1 (negative control): 0.5 mL of distilled water
Group 2 (treated): 0.5 mL of the test substance at 50% in distilled water.
Day 9: occlusive dressing removal
Day 10: Skin reaction evaluation

Rest phase: the animals of both groups were left for 10 d.
Challenge phase: Day 20: the experimental procedure of this phase was identical for both groups,
Group 1 (Negative control) and Group 2 (Treated): to the previously shorn dorso-lumbar zone, an application on either side of the spine, under occlusive dressing for 24 h of- 1 sample cup containing the test substance diluted at 0.5% (MNIC) in distilled water and to the other side of the spine 1 sample cup containing the test substance at 0.25% in distilled water (1/2 MNIC).
Day 21: occlusive dressing removal. A macroscopic evaluation of the cutaneous reactions (erythema and oedema) was conducted and all the local or systemic reactions are recorded as GROUP 1 (Negative control) and
Group 2 (Treated): Day 22 approximately 21 h after removal of the occlusive dressing, the treated zone was shaved, approximately 3 h later, the cutaneous reactions were observed and graded according to the scales, given below. Day 23 24 h later (i.e. 48 h after removal of the occlusive dressing), a second observation was made.
Challenge controls:
The negative control group was treated identically to the treated group.
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde
Positive control results:
The results of the 3 last positive groups, using alpha-Hexylcinnamaldehyde, carried out as method sensibility. Under the experimental conditions, the reference substance, α-Hexylcinnamaldehyde is classified as a skin sensitiser.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.5%
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
Slight erythema in 20% (1/5) of the animals
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No reaction was noted
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.5%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Slight erythema in 1/10 animals
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No reaction noted
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.25%
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
Slight erythema in 1/5 animals
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.25%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No reaction was noted
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.25%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Slight erythema in 10% (1/10)
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No reaction was noted
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
6.25%
No. with + reactions:
3
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
6.25%
No. with + reactions:
1
Total no. in group:
8
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
12.5%
No. with + reactions:
3
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
12.5%
No. with + reactions:
3
Total no. in group:
7
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25%
No. with + reactions:
4
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25%
No. with + reactions:
7
Total no. in group:
24

Results:

Concentrations selected:

Preliminary studies:

MNNC determination:

The results are given in Table 1: No necrosis has been observed for the 2 animals, at the concentration of 0.1 %. Based on this a concentration of 0.1% was selected for intradermal induction in the main study of the Group 2 treated animals.

 

Pre-MNIC determination:

The results are given in Table 2:24 h after the removal of the occlusive dressings, slight to well defined erythema was recorded on the treated area at 50 %, 25 % and 10 % in 2 animals and slight erythema was recorded on the treated area at 5 % in 1 animal.

Based on these results, the concentration selected was 50% for the topical induction of the Group 2 with sodium lauryl sulphate pre-treatment (10% in vaseline) and the MNIC determination began at the concentration 4%.

 

 

MNIC determination:

The results are given in Table 3:Slight to well defined erythema associated or not with a very slight to slight oedema was recorded on the treated area at 4%, 2% and 1%, 24 and 48 h after patch removal. No cutaneous reaction was recorded on the treated areas at 0.5%

Based on these results, the concentrations selected for the challenge phase of the main study were 0.5% (MNIC) and 0.25% (1/2 MNIC)

 

 

Main Study:

Induction phase Group 2: the induction phase was performed by intradermal injection on D0 with the test item at 0.1 % in isotonic sodium chloride and by topical application on D7 with the test item at 50 % in distilled water after treatment with sodium lauryl sulphate 24 h earlier. No cutaneous reactions was recorded after the 1stinduction phase. During the 2ndinduction, dryness was noted in 7 animals (7/10) and a scab was noted in 3 animals (3/10), 24 h after the removal of the occlusive dressing.

 

Induction phase Group 1: the induction phase was performed by intradermal injection on D0 with isotonic sodium chloride and by topical application on D7 with distilled water after treatment with sodium lauryl sulphate 24 h earlier. No cutaneous reaction was recorded after the induction phases.

 

Challenge phase Groups 1 & 2: the test item was diluted at 0.5 % and 0.25 % in distilled water.

 

Sensitising potential assessment

Overall results of the challenge phase with the test item (readings at 24 and 48 h) are given in table 4 Individual scores of macroscopic evaluations performed during challenge phase with the test item are given in table 5.

 

Slight erythema was observed on the area treated with the test item at either 0.5% or 0.25% in 10% (1/10) of Group 2 (treated) animals 24 h after the challenge phase. No reaction was noted in Group 2 (treated) animals treated with test item at either 0.5% or 0.25% at the reading time 48 h.

 

Slight erythema was observed in 10% (1/10) of the animals from the treated group, 24 h after the challenge phase, on the treated area with the test item at 0.25%. No reaction was noted at the reading time 48 h.

Slight erythema was observed in 20% (1/5) of the animals from the negative control group, 24 h after the challenge phase, on the treated area with the test item at 0.25%. No reaction was noted at the reading time 48 h.

 

 

Body Weights:

The body weight of negative control animals (Group 1) and treated animals (Group 2) is presented in tables 6 and 7 respectively. No abnormality was recorded in the body weight gain of either groups.

 

Mortality:

No mortality was registered during the main study.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the substance was not considered to be skin sensitiser in guinea pig.
Executive summary:

A study was conducted to assess the skin sensitising potential of the read-across substance according to OECD Guideline 406 and EU Method B.6, in compliance with GLP. Induction of 10 Guinea Pigs in the treated group (Group 2) with the test substance was by intradermal injection of 0.1% test substance in isotonic sodium chloride and in Freund’s Complete Adjuvant followed one week later by treatment with sodium lauryl sulphate and 24 h thereafter topical application at 50% test substance in distilled water. A negative control group (5 guinea pigs) received intradermal injections of isotonic sodium chloride and Freund’s Complete Adjuvant in isotonic sodium chloride followed one week later by treatment with sodium lauryl sulphate and 24 h thereafter topical application of distilled water. Slight erythema was observed on the area treated with the test substance at either 0.5% or 0.25% in 10% of Group 2 (treated) animals 24 h after the challenge phase. No reaction was noted in Group 2 (treated) animals treated with test substance at either 0.5% or 0.25% at 48 h. Responses observed in the negative and positive control groups were satisfactory. Under the study conditions, the test substance was not considered to be skin sensitiser in guinea pig (Richeux, 2011).

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

In vivo:

A study was conducted to assess the skin sensitising potential of the read across substance, Phosphoric acid, mono- and di-C6-10-alkyl esters, according to OECD Guideline 406 and EU Method B.6, in compliance with GLP. Induction of 10 Guinea Pigs in the treated group (Group 2) with the test substance was by intradermal injection of 0.1% test substance in isotonic sodium chloride and in Freund’s Complete Adjuvant followed one week later by treatment with sodium lauryl sulphate and 24 h thereafter topical application at 50% test substance in distilled water. A negative control group (5 guinea pigs) received intradermal injections of isotonic sodium chloride and Freund’s Complete Adjuvant in isotonic sodium chloride followed one week later by treatment with sodium lauryl sulphate and 24 h thereafter topical application of distilled water. Slight erythema was observed on the area treated with the test substance at either 0.5% or 0.25% in 10% of Group 2 (treated) animals 24 h after the challenge phase. No reaction was noted in Group 2 (treated) animals treated with test substance at either 0.5% or 0.25% at 48 h. Responses observed in the negative and positive control groups were satisfactory. Under the study conditions, the substance was not considered to be skin sensitiser in guinea pig (Richeux, 2011).

Justification for classification or non-classification

Based on the in vivo skin sensitisation study with the read-across substance Phosphoric acid, mono- and di-C6-10-alkyl esters,

, no classification for skin sensitisation is warranted according to EU CLP (1272/2008) criteria.