Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
The experiment was carried out between 21 November 2011 and 06 January 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Read-across study conducted in compliance with agreed protocols, with no deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

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

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
Sponsor's identification: Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts
CAS number : 68140-11-4
Identifier : TIS O2779
Description : Yellow/brown extremely viscous liquid
Batch number : LE 012569
Date received : 20 October 2011
Expiry date : 06 June 2013
Storage conditions: room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Fifteen albino guinea pigs of Dunkin-Hartley strain, supplied by CHARLES RIVER.
- Age at study initiation: 5 weeks old.
- Weight at study initiation: 277 to 340 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): Food (SDS, FD1) was supplied freely.
- Water (e.g. ad libitum): The drinking water (tap water from public distribution system) was supplied freely.
- Acclimation period: Minimum of 5 days, under stabling and nutritional conditions identical to those of the test.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was controlled to remain within target range of 19 - 25°C
- Humidity (%): The relative humidity was controlled to remain within target rahge of 30 to 70%
- Air changes (per hr): The rate of air exchange was approximately fifteeen changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continous light (07.00 to 19.00) and twelve hours darkness.

PREPARATION OF ANIMALS:
The animals were carefully shaved before each test item 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 shaved a second time in this dorso-lumbar zone.
The animals were weighed at the beginning and at the end of the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Remarks:
For the purpose of the study, the test item 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 0.001% in istonic sodium chloride and topical application at 25% in distilled water.
Challenge: 2% and 1% in distilled water
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
For the purpose of the study, the test item 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 0.001% in istonic sodium chloride and topical application at 25% in distilled water.
Challenge: 2% and 1% in distilled water
No. of animals per dose:
Main study:
Group 1 (negative control): 5 animals
Group 2 (treated): 10 animals
Details on study design:
PREPARATION OF TEST ITEM:
For the purpose of the study, the test item was used freshly prepared in isotonic sodium chloride for the intradermal injections and in distilled water for the topical applications. This vehicle was chosen as it produced the most suitable formulation at the required concentration. Indeed, the preparation of the test item at 50% in isotonic sodium chloride (v/v) was a yellow/orange thick cream and the preparation of the test item at 50% in distilled water (v/v) was a yellow/orange thick cream.
Each preparation was done just before the administration was was homogenous (visual determination).

RANGE FINDING TESTS:
Determination by intradermal injection of the Maximum Non Necrotizing Concentration (MNNC):
This test was conducted for the purpose of defining a MNNC of the test item which, on intradermal injection during the induction phase, does not risk causing too great a lesion (non-necrotizingconcentration), should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation.

Two animals received on both sides of the spine, a volume of 0.1 mL of the test item, at 4 concentrations: diluted at 50%, 25%, 10% and 5% in isotonic sodium chloride in view to determine the MNNC.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours 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 item, at 3 concentrations: diluted at 1%, 0.5% and 0.1% in isotonic sodium chloride in view to determine the MNNC.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after injection.

Due to the necrosis registered, the same animal received on both sides of the spine, a volume of 0.1 mL of the test item, at 3 concentrations: diluted at 0.05%, 0.01% and 0.005% in isotonic sodium chloride in view to determine the MNNC.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours 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 item, at 3 concentrations: diluted at 0.001%, 0.0005% and 0.0001% in isotonic sodium chloride in view to determine the MNNC.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after injection.

Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC):
This test evaluated the irritancy potential of the test item to determine whether an application of sodium lauryl sulfate would be needed during topical induction phase.
Four preparations of the test item (50%, 25%, 10% and 5% in isotonic sodium chloride) were applied under occlusive dressing for 24 hours to the shaved dorso-lumbar zone of two guinea pigs.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing.

As one animal was found dead 24 hours after the test item administration, a new animal received the test item in the same experimental conditions, at 3 concentrations: diluted at 25%, 10% and 5% in distilled water in view to determine the MNNC.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours 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 item without risk of an irritant effect during the challenge phase.

Three guinea pigs 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 item placed onto the selected treatment sites at concentrations of 2%, 1%, 0.5% and 0.25% in distilled water and covered with an occlusive dressing for a period of 24 hours.
A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressing.


MAIN STUDY

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

Mortality and clinical signs were recorded daily. The bodyweight of each animal was recorded at the start, after the 2nd induction and at the end of the study.

A. INDUCTION EXPOSURE
Intradermal Induction:
Day 0:
After shearing the scapular zone, three pairs of intradermal injecions (ID) of 0.1 ml were performed on the scapular zone in such a way as an injection on each pair is placed to 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: Freund's Complete Adjuvant at 50% and isotonic sodium chloride

Group 2 (Treated)
- 2 ID: Freund's Complete Adjuvant diluted by 50% in isotonic sodium chloride
- 2 ID: test item at 0.001% in istonic sodium chloride
- 2 ID: a test mixture in equal volumes v/v: Freund's Complete Adjuvant at 50% and the test item at 0.002% in isotonic sodium chloride

Day 1: Skin reaction evaluation

Topical Induction:
Day 8:
A topical application under occlusive dressing for 48 hours was performed on the injection sites of each animal.
GROUP 1 (Negative control)L 0.5 mL of distilled water
GROUP 2 (treated): 0.5 mL of the test item at 25% in distilled water

Day 10: Occlusive dressing removal
Day 11: Skin reaction evaluation

Rest phase: The animals of both groups were left for 10 days.

B. CHALLENGE EXPOSURE
Day 21:
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 hours of:
- sample cup containing the test item diluted at 2% (MNIC) and to the other side of the spine 1 sample cup containing the test item diluted at 1% (1/2 MNIC)

Day 22: Occlusive dressing removed.

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 23:
Approximately 21 hours after removal of the occlusive dressing, the treated zone was shaved. Approximately 3 hours later, the cutaneous reactions were observed and graded according to the scales given below:

Grading scales:
Erythema:
0: No visible modification
1: Slight or patches of erythema
2: Moderate confluent erythema
3: Internal erythema and swelling

Oedema:
0: No visible modification
1: Slight oedema
2: Moderate oedema
3: Important oedema

Day 24: 24 hours later (i.e. 48 hours after removal of the occlusive dressing), a second observation was made.

Day 25: 48 hours later (i.e. 72 hours after removal of the occlusive dressing), a third observation was made.


OTHER:
Interpretation of reactions:
All the animals with scores (erythema or oedema) above or equal to 1 during the challenge phase, were considered positive.
The percentage of animals that showed a contact sensitivity potential were calculated from the readings at 24 and 48 hours.

A comparison of the intensities and persistence of reactions at the test item challenge sites in the test and control animals permits identification of sensitisation reactions.

If the test item at the maximum non-irritant concentration produces reactions in treated group animals at the 24 or 48-hour readings, these reactions were attributed to skin sensitisation. This pre-supposes that no similar reactions were observed in the test item challenge sites of any of the
control group animals. If irritation was observed in the control group animals, only reactions in the treated group animals that exceed the most severe reaction seen in the control group animals are attributed to skin sensitisation. The results were expressed in terms of incidence and severity
of responses, i.e.:

Incidence Score: The number of treated group animals showing skin reactions greater than the most severe reaction observed in the control group animals, expressed as a fraction of the total number of test group animals.
Severity Score: The sum of the values assigned to the erythematous skin responses at the test item challenge sites of the test and control group animals, at each evaluation, divided by the number of animals in that group.























Challenge controls:
The negative control group was treated identically to the treated group.
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:
The results of the 3 latest positive control groups using alpha-Hexylcinnamaldehyde are attached (see attached background material). Under the experimental conditions, the reference substance, alpha-Hexylcinnamaldehyde, is classified as a skin sensitiser.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 2%. No with. + reactions: 8.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
2%
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2%. No with. + reactions: 7.0. Total no. in groups: 10.0.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
2%
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 2%. No with. + reactions: 6.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
1%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
-
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: -. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
-
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: -. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
-
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: -. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Results

Concentrations selected

Preliminary studies:

- MNNC determination:

The results are given in Table 1 (see attached background material).

No necrosis was observed at a concentration of 0.001%. Based on this a concentration of 0.001% 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 (see attached background material).

24 hours after the removal of the occlusive dressings, well defined to severe erythema was noted on the treated areas at 25% and 50% in the first animal and well defined erythema was noted on the treated areas at 25%, 10% and 5% in the second animal.

Based on these results, the concentration selected was 25% for the topical induction of the Group 2 and MNIC determination began at the concentration of 2%.

Furthermore, an application of sodium lauryl sulfate would be not needed 24 hours before the topical induction.

- MNIC determination:

The results are given in Table 3 (see attached background material).

No cutaneous reaction was observed on any of the treated areas at 24 and 48 hours after the removal of the occlusive dressings.

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

Main study:

- Induction phase Group 1 (Negative control):

The induction phase was performed by intradermal injection on D0 with isotonic sodium chloride and by topical application on D7 with distilled water.

No cutaneous reaction was recorded after the intradermal induction. After topical nduction, dryness was noted in 5 animals (5/5), 24 hours after the removal of the occlusive dressing.

- Induction phase Group 2 (Treated):

The induction phase was performed by intradermal injection on D0 with the test item at 0.001% in isotonic sodium chloride and by topical application on D7 with the test item at 25% in distilled water. No cutaneous reaction was recorded after the intradermal induction. After topical induction, dryness was noted in 10 animals (10/10), 24 hours after the removal of the occlusive dressing.

- Challenge phase Groups 1 & 2:

The test item was used diluted at 2% and at 1% in distilled water.

Sensitising potential assessment:

Overall results of the challenge phase with the test item (readings at 24, 48 and 72 hours) are given in Table 4 (see attached background material).

Individual scores of macroscopic evaluations performed during challenge phase with the test item are given in Table 5 (see attached background material).

Slight to moderate erythema was observed on the area treated with the test item at 2% in 80% (8/10), in 70% (7/10) and in 60% (6/10) of Group 2 (treated) animals at 24, 48 and 72 hours after the challenge phase respectively. Slight oedema was observed on the area treated with the test item at 2% in 30% (3/10) of Group 2 (treated) animals at 24 hours after the challenge phase.

No cutaneous intolerance reaction was observed on the area treated with the test item at 2% in animals from Group 1 (negative control) after the challenge phase.

Slight erythema was observed on the area treated with the test item at 1% in 20% (2/10) of Group 2 (treated) animals at 24 and 48 hours after the challenge phase. No cutaneous reactions were noted 72 hours after the challenge phase.

No cutaneous intolerance reaction was observed on the area treated with the test item at 1% in animals from Group 1 (negative control) after the challenge phase.

Weight evolution:

The weight growth of negative control animals (Group 1) and treated animals (Group 2) is presented in Tables 6 and 7 (see attached background material) respectively.

No abnormality was recorded in the body weight gain of both groups.

Mortality:

No mortality was registered during the main test.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In view of the results, under these experimental conditions, the test item Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts, is a skin sensitiser.
Executive summary:

The aim of the study was to evaluate the possible cutaneous allergenic activity of the test item after intradermal and topical administration in guinea pigs. The experimental protocol was established according the OECD guideline No. 406 dated July 17th, 1992 and the test method B.6 of the council regulation No. 440/2008.

Induction of 10 Guinea Pigs in the treated group with the test item Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts was by intradermal injections with 0.001% test item in isotonic sodium chloride and in an emulsion of Freund's Complete followed one week later by topical application at 25% test item in distilled water. A negative control group (5 guinea pigs) received intradermal injections of isotonic sodium chloride and an emulsion of Freund’s Complete Adjuvant at 50% / isotonic sodium chloride Freund's Complete followed one week later by topical application of distilled water. After a 10-day rest phase, the treated group and negative control group were challenged with a single topical application of the test item diluted at 2% and 1% in distilled water under an occlusive dressing for 24 hours.

Slight to moderate erythema was observed on the area treated with the test item at 2% in 80% (8/10), in 70% (7/10) and in 60% (6/10) of Group 2 (treated) animals at 24, 48 and 72 hours after the challenge phase respectively. Slight oedema was observed on the area treated with the test item at 2% in 30% (3/10) of Group 2 (treated) animals at 24 hours after the challenge phase.

No cutaneous intolerance reaction was observed on the area treated with the test item at 2% in animals from Group 1 (negative control) after the challenge phase.

Slight erythema was observed on the area treated with the test item at 1% in 20% (2/10) of Group 2 (treated) animals at 24 and 48 hours after the challenge phase. No cutaneous reactions were noted 72 hours after the challenge phase.

No cutaneous intolerance reaction was observed on the area treated with the test item at 1% in animals from Group 1 (negative control) after the challenge phase. In view of the results, under these experimental conditions, the test item Fatty acids, C18 unsaturated, reaction products with diethylenetriamine, acetate salts, is a skin sensitiser.