Registration Dossier

Administrative data

Description of key information

sensitising, Guinea Pig (female), eq. to OECD TG 406, 2015

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12/2014 - 04/2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP; minor deviations (minimum relative humidity) not considered to impact the reliability of the study. Scientific justification is attached by the applicant.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
Deviations from the minimum daily mean relative humidity occurred. Based on HCD this was not considered to impact the reliability of the study.
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
See above.
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Remarks:
See above.
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147 (2000)
Deviations:
no
Remarks:
See above.
Principles of method if other than guideline:
Method employed in this study for the detection of delayed contact hypersensitivity was the guinea-pig maximization test described by B. Magnusson and A.M. Kligman (1970) in "Allergic Contact Dermatitis in the Guinea-Pig: Identification of contact allergens"; C.C. Thomas, USA.
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2013; signature: May 2013
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Scientific Justification for completion of GPMT to fulfil REACH Regulation (EC) 1907/2006: Annex VII - 8.3 Information Requirement is attached
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: Young adult (approximately 4 weeks old).
- Weight at study initiation: 250 – 282 g (mean weight: 266 g).
- Housing: Group housing of maximally 5 animals per labelled Noryl cages containing sterilized sawdust as bedding material, shelters and play tunnels as cage enrichment.
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (details in the full study report). In addition, hay was provided at least twice a week.
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 – 70%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: To: 10-02-2015 to 08-04-2015
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Preliminary irritation testing: A series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1%
Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 2%
- Topical: 10%
- Challenge: 10%
% are percent v/v of test substance in vehicle. Vehicle: corn oil, was chosen on the basis of maximising the solubility of the test substance; following evaluation of test item data and trial formulation results. The vehicle was selected on this basis from (in order of preference): Water; 1%; Aqueous carboxymethyl cellulose; Corn oil; Propylene glycol; Polyethylene glycol 400.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Preliminary irritation testing: A series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1%
Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 2%
- Topical: 10%
- Challenge: 10%
% are percent v/v of test substance in vehicle. Vehicle: corn oil, was chosen on the basis of maximising the solubility of the test substance; following evaluation of test item data and trial formulation results. The vehicle was selected on this basis from (in order of preference): Water; 1%; Aqueous carboxymethyl cellulose; Corn oil; Propylene glycol; Polyethylene glycol 400.
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Test group: 10; Control group: 5
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. Intradermal Injection: A series of four test substance concentrations was tested, the highest concentration being the maximum concentration that could technically be injected. Two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment. Based on the results in the initially treated animals, two additional animals were treated in a similar manner with four lower concentrations at a later stage. Epidermal application: A series of four test substance concentrations was tested, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on medical tape and held in place with micropore tape and elastic bandage. The resulting dermal reactions were assessed for irritation 24 and 48 hours after exposure. Based on the results in the initially treated animals, one additional animal was treated in a similar manner with two lower concentrations at a later stage.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 1 intradermal induction and Day 8 epidermal induction (topical booster)
- Test groups: Test substance in 1:1 mixture FCA; 2% test item intradermal induction and additional 10% epidermal induction.
- Control group: Vehicle and FCA only.
- Site: intradermal induction – three pairs of injections in clipped scapular region;
- Frequency of applications: Not applicable.
- Duration: 0-8 days. On day 8 - 48 hours for epidermal induction. The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.
- Concentrations: Intradermal induction: A) A 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection; B) The test substance at a 2% concentration ; C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant. Epidermal induction: The scapular area between the injection sites was clipped and subsequently treated with 0.5 mL of a 10% test substance concentration using occlusive dressing.
The control group were treated as described for the experimental group except that, instead of the test substance, the vehicle was administered.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 24 hours (epidermal challenge)
- Exposure period: Day 23 the dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
- Test groups: 1; test substance 10% in vehicle.
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 10% using occlusive dressing.
- Evaluation (hr after challenge): 24 and 48 hours after dressing removal (at Day 23 and 24).
The control group were treated as described for the experimental group except that, instead of the test substance, the vehicle was administered.

OTHER: Mortality, toxicity and body weights along with irritation were examined as part of the study.
Challenge controls:
(Naive) negative control groups consisting of 5 females were exposed to the vehicle in the induction and challenge, consistent the main study with the difference that instead of test substance only the vehicle was administered.
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamicaldehyde (20%)
Positive control results:
A reliability check was performed (within 6 months of the study) to check the sensitivity of the test system and the reliability of the experimental techniques used. The study used the same conditions as the main study using Alpha-Hexylcinnamicaldehyde (20%) as positive control. Test substance concentrations selected for this study were: Intradermal induction: A 20% solution in water (w/w); Epidermal induction: 100% (undiluted) and Challenge: a 20% solution in water (w/w).
The skin reactions observed in seven experimental animals in response to the 20% test substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 90% to the 20%w/w concentration.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Scaliness was reported in 3/10 and scaps in 2/10 (Scabs: was inconjunciton to Score =1 in 1/10); maximum score = 1 (Discrete or patch erythema)
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Scaliness was reported in 3/10 and scaps in 2/10 (Scabs: was inconjunciton to Score =1 in 1/10); maximum score = 1 (Discrete or patch erythema).
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
Superficial necrosis was reported in 5/10 and scalines in 5/10 (which was not considered indicative of sensitisation).
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: Superficial necrosis was reported in 5/10 and scalines in 5/10 (which was not considered indicative of sensitisation)..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None reported; maximum score = 0 (no visible change)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None reported; maximum score = 0 (no visible change).
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
maximum score = 0 (no visible change); scalines in 3/5 (which was not considered indicative of sensitisation).
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: maximum score = 0 (no visible change); scalines in 3/5 (which was not considered indicative of sensitisation)..
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
non-concurrent: reliability check conducted (documented in the full study report)
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
20%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
non-concurrent: reliability check conducted (documented in the full study report)
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study used, the test material is considered to be a contact sensitizer.
Executive summary:

The study was performed according to a method equivalent to guideline OECD TG 406 EU Method B.6 and EPA OPPTS 870.2600 and JMAFF Guidelines and consistent with Magnusson-Kligman Guinea Pig Maximisation test to assess the skin sensitisation potential of the test substance. The choice of vehicle (corn oil) was determined based on solubility testing conducted within the study. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with a 2% concentration and epidermally exposed to a 10% concentration. Five control animals were similarly treated, but with vehicle alone (corn oil). Two weeks after the epidermal application all animals were epidermally challenged with a 10% test substance concentration and the vehicle. In the challenge phase, signs of superficial necrosis were observed in five experimental animals in response to the 10% test substance concentration after 48 hours. Furthermore, skin reactions of grade 1, were observed in one experimental animal after 24 hours. No erythema or signs of necrosis were evident in the control animals. Scaliness was seen in some treated skin sites of the experimental and control animals. The skin reactions other than scaliness observed in response to a 10% test substance concentration in five (out of ten) 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 50 per cent. Under the conditions of this study, the test substance is considered to be a contact skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Key data : OECD TG 406, 2015 : The study was performed according to a method equivalent to guideline OECD TG 406 EU Method B.6 and EPA OPPTS 870.2600 and JMAFF Guidelines and consistent with Magnusson-Kligman Guinea Pig Maximisation test to assess the skin sensitisation potential of the test substance. The choice of vehicle (corn oil) was determined based on solubility testing conducted within the study. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with a 2% concentration and epidermally exposed to a 10% concentration. Five control animals were similarly treated, but with vehicle alone (corn oil). Two weeks after the epidermal application all animals were epidermally challenged with a 10% test substance concentration and the vehicle. In the challenge phase, signs of superficial necrosis were observed in five experimental animals in response to the 10% test substance concentration after 48 hours. Furthermore, skin reactions of grade 1, were observed in one experimental animal after 24 hours. No erythema or signs of necrosis were evident in the control animals. Scaliness was seen in some treated skin sites of the experimental and control animals. The skin reactions other than scaliness observed in response to a 10% test substance concentration in five (out of ten) 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 50 per cent. Under the conditions of this study, the test substance is considered to be a contact skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance does meet classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation (category 1B): H317

In the GPMT test the substance produced a sensitisation rate of 5/10 in a test consistent with established adjuvant-test methodology. Under EU criteria significant effects were observed in the in vivo GPMT study on exposure to the substance at > 1% v/v intradermal induction dose. On the basis of this study it is suggested that the test substance meets the Regulation (EC) 1272/2008: Skin Sensitisation category 1B (H317) classification.