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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experiment was carried out between 19 September 2011 and 21 October 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

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
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Reference substance name:
Esterification products of Fatty acids C18 unsaturated and triethanolamine
EC Number:
939-649-8
Cas Number:
1474044-69-3
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
IUPAC Name:
Esterification products of Fatty acids C18 unsaturated and triethanolamine
Test material form:
liquid: viscous

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: 4 weeks old
- Weight at study initiation: 231 g - 290 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 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 shorn a second time in this dorso-lumbar zone.
The animals were weighed at the beginning, after the 2nd induction and at the end of the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: The test item was used freshly prepared in olive oil for the intradermal injections and in liquid paraffin for the topical applications.
Concentration / amount:
Main study:
Induction:
Intradermal injection - 10% test item in olive oil
Topical application - 50% test item in liquid paraffin

Challenge: Test item diluted at 6.25% and 3.125% in liquid paraffin.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: The test item was used freshly prepared in olive oil for the intradermal injections and in liquid paraffin for the topical applications.
Concentration / amount:
Main study:
Induction:
Intradermal injection - 10% test item in olive oil
Topical application - 50% test item in liquid paraffin

Challenge: Test item diluted at 6.25% and 3.125% in liquid paraffin.
No. of animals per dose:
Group 1 (negative control): 5
Group 2 (treated): 10
Details on study design:
PRELIMINARY STUDIES:

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 item 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 item, at 4 concentrations: diluted at 50%, 25%, 10% and 5% in olive oil in order to determine the MNNC.
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 item, at 2 concentrations: diluted at 10% and 5% in olive oil in order to determine the MNNC. As necrosis was noted in the first animal with the tested concentrations 50% and 25%, these two concentrations were not tested in the second animal.
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 the topical induction phase.
Four preparations of the test item (50%, 25%, 10% and 5% in liquid paraffin) were applied under occlusive dressing for 24 hours to the shaved dorso-lumbar zone of one guinea pig.
A macroscopic evaluation of the cutaneous reactions was conducted 24 hours after removal of the dressing.
The results were confirmed with a new animal. The animal received the test item in the same experimental conditions, at 4 different concentrations: diluted at 50%, 25%, 10% and 5% in liquid paraffin.
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. olive oil and liquid paraffin).
During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites at concentrations of 25%, 12.5%, 6.25% and 3.125% in liquid paraffin 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 dressing.

MAIN STUDY:
Group 1 (negative control): 5 female guinea pigs
Group 2 (treated): 10 female guinea pigs

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.

INDUCTION PHASE:
1st INTRADERMAL INDUCTION:
Day 0: After shearing the scapular zone, three (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: olive oil
- 2 ID: a mixture with equal volumes v/v: Freund's Complete Adjuvant at 50% and olive oil

Group 2 (Treated):
- 2 ID: Freund's Complete Adjuvant diluted by 50% in isotonic sodium chloride
- 2 ID: test item at 10% in olive oil
- 2 ID: a test mixture in equal volumes v/v: Freund's Complete Adjuvant at 50% and the test item at 20% in olive oil.

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 sulfate at 10% in thick Vaseline, in order to create a local irritation.

Day 7: A topical application under occlusive dressing for 48 hours 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 liquid paraffin
Group 2 (treated): 0.5 mL of the test item at 50% in liquid paraffin.

Day 9: Occlusive dressing removal

Day 10: Skin reaction evaluation

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

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 hours of:
- 1 sample cup containing the test item diluted at 6.25% (MNIC) in liquid paraffin and to the other side of the spine 1 sample cup containing the test item diluted at 3.125% in liquid paraffin (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 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. (see any other information on materials and methods incl .tables)

Day 23: 24 hours later (i.e. 48 hours after removal of the occlusive dressing), a second observation was made.

Day 24: 48 hours later (i.e. 72 hours after removal of the occlusive dressing), a third observation was made.





































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 last positive groups, using alpha-Hexylcinnamaldehyde, carried out as method sensibility are attached (see attached background material). Under the experimental conditions, the reference substance, α-Hexylcinnamaldehyde is classified as a skin sensitiser.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
6.25%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Slight erythema observed in 10% (1/10) animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 6.25%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Slight erythema observed in 10% (1/10) animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
6.25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No reaction noted
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 6.25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No reaction noted.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
6.25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No reaction noted
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 6.25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No reaction noted.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
6.25%
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: 6.25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
6.25%
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: 6.25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
6.25%
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: 6.25%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
3.125%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Slight erythema observed in 10% (1/10) animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 3.125%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Slight erythema observed in 10% (1/10) animals .
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
3.125%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Slight erythema observed in 10% (1/10) animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 3.125%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Slight erythema observed in 10% (1/10) animals.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
3.125%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No reaction noted
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 3.125%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No reaction noted.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
3.125%
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: 3.125%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
3.125%
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: 3.125%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
3.125%
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: 3.125%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Concentrations selected

Preliminary studies:

- MNNC determination:

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

No necrosis was observed for the two animals at a concentration of 10%. Based on these results, a concentration of 10% 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, slight erythema was recorded on the treated area at 50% in one 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 of 25%.

- MNIC determination:

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

Well defined erythema and slight erythema was recorded on the treated area at 25% in one animal at 24 and 48 hours respectively after the patch removal. Slight erythema was recorded 24 hours after the patch removal on the treated area at 12.5% in a further animal. No other cutaneous reactions were observed at any concentration in any animals at either 24 and 48 hours after removal of the occlusive dressings.

Based on these results, the concentrations selected for the challenge phase of the main study were 6.25% (MNIC) and 3.125% (1/2 MNIC).

Main Study:

- Induction phase Group 2:

The induction phase was performed by intradermal injection on D0 with the test item at 10% in olive oil and by topical application on D7 with the test item at 50% in liquid paraffin after treatment with sodium lauryl sulphate 24 hours earlier.

No cutaneous reaction was recorded after the intradermal induction. After topical induction, dryness was noted in 6 animals (6/10), 24 hours after the removal of the occlusive dressing.

- Induction phase Group 1:

The induction phase was performed by intradermal injection on D0 with olive oil and by topical application on D7 with liquid paraffin after treatment with sodium lauryl sulphate 24 hours earlier.

No cutaneous reaction was recorded after intradermal induction. After topical induction, dryness was noted in 2 animals (2/5), 24 hours after the removal of the occlusive dressing.

Challenge phase Groups 1 and 2:

The test item was used diluted at 6.25% and 3.125% in liquid paraffin.

Sensitising potential assessment

Overall results of the challenge phase with the test item for Group 1 (negative control) and Group 2 (treated) 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 for Group 1 (negative control) and Group 2 (treated) at 24, 48 and 72 hours are given in table 5 (see attached background material).

Slight erythema was observed on the area treated with test item at 6.25% in 10% (1/10) of Group 2 (treated) animals at 24 hours after the challenge phase. No reaction was noted at the reading time of 48 or 72 hours.

No cutaneous intolerance reaction was recorded on the area treated with test item at 6.25% in animals from Group 1 (negative control) after the challenge phase.

Slight erythema was observed on the area treated with test item at 3.125% in 10% (1/10) of Group 2 (treated) animals 24 and 48 hours after the challenge phase. No reaction was noted at the reading time of 72 hours.

No cutaneous intolerance reaction was recorded on the area treated with test item at 3.125% in animals from Group 1 (negative control) after the challenge phase.

Body weight:

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

No abnormality was recorded in the body weight gain of either group.

Mortality:

No mortality was registered during the main test.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the substance was found not to be 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 accordingthe OECD guideline No. 406 dated July 17th, 1992 and the test method B.6 of the councilregulation No. 440/2008.

Induction of 10 Guinea Pigs in the treated group (Group 2) with the test item was by intradermal injections with 10% test item in olive oil and in Freud’s Complete Adjuvant followed one week later by treatment with sodium lauryl sulphate and 24 hours thereafter topical application at 50% test item in liquid paraffin. A negative control group (5 guinea pigs) received intradermal injections of olive oil, Freund’s Complete Adjuvant at 50% in olive oil and Freund’s Complete Adjuvant at 50% in isotonic sodium chloride followed one week later by treatment with sodium lauryl sulfate and 24 hours thereafter topical application of liquid paraffin. 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 6.25% and 3.125% in liquid paraffin under an occlusive dressing for 24 hours.

Slight erythema was observed on the area treated with test item at 6.25% in 10% (1/10) of Group 2 (treated) animals at 24 hours after the challenge phase. No reaction was noted at the reading time of 48 or 72 hours.

No cutaneous intolerance reaction was recorded on the area treated with test item at 6.25% in animals from Group 1 (negative control) after the challenge phase.

Slight erythema was observed on the area treated with test item at 3.125% in 10% (1/10) of Group 2 (treated) animals 24 and 48 hours after the challenge phase. No reaction was noted at the reading time of 72 hours.

No cutaneous intolerance reaction was recorded on the area treated with test item at 3.125% in animals from Group 1 (negative control) after the challenge phase.

In conclusion, in view of these results, under these experimental conditions, the test item is not a skin sensitiser.