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Administrative data

Description of key information

non-sensitising, Guinea Pig (female), OECD TG 406, 2016

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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. Information as to the availability of the study is attached by the applicant.
Justification for type of information:
Information as to the availability of the in vivo study is provided in 'attached justification'.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
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 - "The identification of contact allergens by animal assay, the guinea pig maximisation method"; Invest. Dermatol. 1969. 52, 268-276
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147 (2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: April 2015; signature: October 2015
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Scientific justification and information as to the availability of the study is attached by the applicant in 'attached background material'.
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 3- 4 weeks old).
- Weight at study initiation: 260 – 284 g (mean weight: 271.1 g).
- Housing: Group housing of 3 per labelled polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted with a stainless steel lid.
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (details in the full study report).
- 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; identical to the test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 – 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: 02-05-2016 to 27-05-2016
Route:
intradermal
Vehicle:
olive oil
Concentration / amount:
- Intradermal: 10% test material in Olive Oil
Day(s)/duration:
Day 1
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
epicutaneous, occlusive
Concentration / amount:
- Topical: 100% test material (undiluted)
Day(s)/duration:
Day 8
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Remarks:
olive oil for the intradermal injections and in liquid paraffin for the topical applications
Concentration / amount:
- Challenge: 100% in liquid paraffin
Day(s)/duration:
Day 21
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. Pre-MNNC: Two received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 concentrations: undiluted (100%) and diluted at 50%, 20% and 10% in olive oil in view to determine the MNNC with macroscopic evaluation at 24 hours. PreMNIC: Two were treated with test item was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 hours, at 4 different concentrations: undiluted (100%) and diluted at 50%, 20% and 10% in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after dressing removal. MNIC: This test was carried out for the purpose of determining the MNIC of the test item without risk of an irritant effect during the challenge phase. Three were treated according to the same treatment as animals from GROUP 1 (control) for the induction phase (i.e. olive oil and liquid paraffin). During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 hours at 2 different concentrations: undiluted (100%v/v) and 50%v/v in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after dressing removal.

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%. This determined the Maximal Non Necrotizing Concentration (MNNC); Pre-Maximal Non Irritant Concentration (Pre-MNIC) and the Maximal Non Irritant Concentration (MNIC) respectively.
Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 10% test material in Olive Oil
- Topical: 100% test material (undiluted)
- Challenge: 100% in liquid paraffin
% are percent v/v of test substance in vehicle. Vehicle: Olive oil (intradermal induction) and liquid paraffin (topical), was chosen on the basis of the most suitable formulation at the required concentrations and maximising the solubility of the test substance. The preparation of the test item at 50% in physiological solution (v/v) was non-homogenous with two phases, within in pre-testing solubility testing.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 0 intradermal induction and Day 8 topical induction (topical booster)
- Test groups: duplicate injections as follows: 2 ID: Freund's Complete Adjuvant diluted at 50% in olive oil; 2 ID: test item at 10% in olive oil ; 2 ID: a test mixture in equal volumes v/v of FCA at 50% and the test item at 20% in olive oil
- Control group: Vehicle and FCA only.
- Site: intradermal induction – three pairs of injections in clipped scapular region;
- Frequency of applications:
- Duration: 0-7 days. On day 7, shorn and SLS at 10% application. On day 8 - 48 hours for epidermal induction. The dressing was removed after 48 hours exposure
- Concentrations: Intradermal induction: A) A 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection; B) The test substance at a 10% concentration ; C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant. Topical induction: The scapular area between the injection sites was clipped and brushed with a solution of SLS at 10%. Then the following day subsequently treated with 0.5 mL of a 100% 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 (topical challenge)
- Exposure period: Day 21 the dressing was removed after 24 hours exposure. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
- Test groups: 1; test substance 100% and 50% in liquid paraffin vehicle.
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 100 and 50% 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 during induction.
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamicaldehyde (20%)
Positive control results:
A reliability check was performed (three tests are listed at 6, 12 and 18 months of the study and presented in the full study report) 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 (at 6.25 and 12.5 concentrations %v/v) as positive control.
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 80 - 100% to the 12.5 %w/w concentration.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Total no. in groups: 10.0. Clinical observations: None reported; maximum score = 0 (no visible change).
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Total no. in groups: 10.0. Clinical observations: None reported; maximum score = 0 (no visible change).
Remarks on result:
no indication of skin 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:
Total no. in groups: 5.0. Clinical observations: None reported; maximum score = 0 (no visible change).
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Total no. in groups: 5.0. Clinical observations: None reported; maximum score = 0 (no visible change).
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
12.5%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
non-concurrent PC conducted within 6 months of the definitive test; % sensitised was based on comparison of challenge sites with test and controls.

Table 1: Preliminary irritation study

Skin reactions after intradermal injection (24 hours)

Number

100%v/v

50%v/v

20%v/v

10%v/v

1

Ne

Ne

Ne

0

2

Ne

Ne

Ne

0

 

 

 

 

 

Ne = Necrosis

0 = No visible change

MNNC: 10% in olive oil

 

Table 2: Preliminary irritation study

Skin reactions after topical application (24 hours)

Number

100%v/v

50%v/v

20%v/v

10%v/v

1

0

0

0

0

2

0

0

0

0

 

 

 

 

 

0 = No visible change

Pre-MNIC: 100%

 

Table 3. Preliminary irritation study

Number

Hours

100%v/v

50%v/v

20%v/v

10%v/v

1

24

0

0

0

0

 

48

0

0

0

0

2

24

0

0

0

0

 

48

0

0

0

0

3

24

0

0

0

0

 

48

0

0

0

0

 

 

 

 

 

 

MNIC: 100% in liquid paraffin

 

Table 4.

 

 

20% MNIC

 

Liquid Paraffin

 

 

Number

24 hours

48 hours

24 hours

48 hours

Control

1

0

0

0

0

 

2

0

0

0

0

 

3

0

0

0

0

 

4

0

0

0

0

 

5

0

0

0

0

Test group

1

0

0

0

0

 

2

0

0

0

0

 

3

0

0

0

0

 

4

0

0

0

0

 

5

0

0

0

0

 

6

0

0

0

0

 

7

0

0

0

0

 

8

0

0

0

0

 

9

0

0

0

0

 

10

0

0

0

0

Where:

0 = No visible change

1 = Discrete or patchy erythema

2 = Moderate and confluent erythema

3 = Intense erythema and swelling

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the test material is not considered to be a contact sensitizer.
Executive summary:

The study was performed according to a method equivalent to guideline OECD TG 406 and EU Method B.6 and consistent with Magnusson-Kligman Guinea Pig Maximisation test under GLP to assess the skin sensitisation potential of the test substance. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 10% and topical application at 100%) and a ten day rest phase of ten experimental animals were challenged with 100% (undiluted) test item and 50%v/v in liquid paraffin along with parallel control challenged at 100% (undiluted) test item and 50%v/v in liquid paraffin in five animals. The treated group (treatment dose of 100%v/v and/or 50%v/v in liquid paraffin), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase at 24 or 48 hours. In the control group (associated with the treatment dose of 100%v/v and/or 50%v/v in liquid paraffin), no cutaneous intolerance reaction was recorded at 24 and 48 hours. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (control item). Under the conditions of this study, the test substance is not considered to be a contact skin sensitizer.

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

Key study: OECD TG 406, 2016 : The study was performed according to a method equivalent to guideline OECD TG 406 and EU Method B.6 and consistent with Magnusson-Kligman Guinea Pig Maximisation test under GLP to assess the skin sensitisation potential of the test substance. Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 10% and topical application at 100%) and a ten day rest phase of ten experimental animals were challenged with 100% (undiluted) test item and 50%v/v in liquid paraffin along with parallel control challenged at 100% (undiluted) test item and 50%v/v in liquid paraffin in five animals. The treated group (treatment dose of 100%v/v and/or 50%v/v in liquid paraffin), no macroscopic cutaneous reactions attributable to allergy were noted after the challenge phase at 24 or 48 hours. In the control group (associated with the treatment dose of 100%v/v and/or 50%v/v in liquid paraffin), no cutaneous intolerance reaction was recorded at 24 and 48 hours. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin (control item). Under the conditions of this study, the test substance is not considered to be a contact skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Full information as to the availability of the study is provided by the applicant. The applicant conducted appropriate in silico assessment using appropriate computational models prior to testing and there was an absence of known skin sensitisation protein binding alerts on associated main constituents. There were pre-hapten alerts (after autoxidation) on traces/impurities (out of domain). Appropriate in chemico or in vitro methods were not available due to limited test assay domains of respective methods when applied to the substance.

 

In the GPMT test the severity of effects were non-existent (score = 0) at 24 and 48 hours in ten organisms when topically challenged at up to the maximum non-irritating concentration 100%v/v and 50%v/v (liquid paraffin) following intradermal induction at 10%v/v (olive oil) with topical booster at 100%. The test was consistent with established adjuvant-test methodology. Under EU criteria there was no or only very limited effects observed in the in vivo GPMT study on exposure to the substance at > 1% v/v intradermal induction dose inadequate for classification. The substance cannot be considered a skin sensitiser as there is no evidence of significant effects in available animal studies and no history of the test item yielding positive responses in humans.

 

The overall weight of evidence is indicative of the substance not meeting the relevant classification criteria.

 

References:

1. ECHA Guidance on Application on the CLP Criteria, section 3.4.2.2 (v5.0, July 2017)