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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:
July 15, 2015 to August 14, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 406 and EU Method B.6 in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

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:
Fatty acids, tall-oil, reaction products with triethylenetetramine
EC Number:
272-905-0
EC Name:
Fatty acids, tall-oil, reaction products with triethylenetetramine
Cas Number:
68919-79-9
Molecular formula:
Not applicable for this UVCB.
IUPAC Name:
Fatty acids, tall-oil, reaction products with triethylenetetramine
Test material form:
semi-solid (amorphous): gel
Remarks:
migrated information: paste

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
- Age and weight range (at order): 4 to 5 weeks of age; 250 to 300 grams
- Supplier: Charles River Italia S.p.A., Calco (Lecco), Italy
- Breeder: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’arbresle Cedex, France
- Date of arrival: July 02, 2015
- Weight range at arrival: 251 to 274 grams
- Acclimatisation period: At least 5 d
- Veterinary health check: During acclimatisation period
- Identification: Permanent by tattoo on the ear, following randomisation at arrival
- Animals per cage: Up to 5
- Housing: Noryl cages measuring 74.354.325 cm
- Cage control: Daily inspected and changed as necessary (at least 3 times/week)
- Water: Drinking water supplied to each cage via a water bottle
- Water supply: Ad libitum
- Diet: 8GP17
- Diet supply: Ad libitum throughout the study
- Room lighting: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 h
- Air changes Approximately 15 to 25 air changes/h
- Temperature range: 22°C±2°C
- Relative humidity range: 55%±15%

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Corn oil and 50% v/v emulsion of Freund’s complete adjuvant (FCA) in sterile water
Concentration / amount:
Induction by intradermal injection (tolerated by the test system): 0.5% in FCA or corn oil
Induction by topical application: 50% in corn oil
Challenge phase: 20% in corn oil
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Corn oil and 50% v/v emulsion of Freund’s complete adjuvant (FCA) in sterile water
Concentration / amount:
Induction by intradermal injection (tolerated by the test system): 0.5% in FCA or corn oil
Induction by topical application: 50% in corn oil
Challenge phase: 20% in corn oil
No. of animals per dose:
Number of animals in test group: 10
Number of animals in control group: 5
Details on study design:
Experimental design
The study was divided into 2 distinct phases. The first of these was a dose-ranging screen which was used to determine suitable dose levels for use in the second phase. This second phase corresponded to the main study: determination of the sensitisation potential of the test substance.

Intradermal injection tolerance test
- Allocation
One group of 2 animals was allocated to the intradermal injection tolerance test.
- Preparation of the site (Day 1)
The hair over the scapulae was removed in the allocated animals using an electric clipper with suitable blade.
- Dosages
Six sites were selected on each animal over the shaved scapulae. Each site was injected with a single concentration of the test substance. Concentrations of 50, 20, 10, 5, 1 and 0.5% in corn oil were selected.
- Dosing method
On Day 1, a single intradermal injection of 0.1 mL of each concentration investigated was administered with a graduated syringe.
- Observations
Observation of the treated sites was performed on Day 2 (approximately 24 h after dosing) and on Day 6 for any signs of reaction to treatment according to the Draize scoring scale below:

Reaction to treatment Value
No erythema 0
Very slight erythema 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema 4

Topical application tolerance test
- Allocation
One group of 5 animals was allocated to the topical application tolerance test.
- Preparation of the site (Day 1)
The hair over the scapulae was removed using an electric clipper with suitable blade. Each animal was then injected intradermally at the prepared site with two injections, each of 0.1 mL, of emulsified Freund’s complete adjuvant.
- Dosing
7 d after the preparation of the test site, the flanks of each animal were clipped free of hair. Each animal was dosed with 2 concentrations of the test substance, 1 on each flank such that a total of 5 concentrations (50, 20, 10, 5 and 1% in corn oil) of the test substance were each dosed in duplicate. A gauze patch measuring 20x20 mm was soaked with 0.2 mL of the test substance and placed onto the selected treatment site. When both sites of the animal had been treated, they were covered with a strip of synthetic film and the trunk wrapped with an elastic adhesive bandage to maintain the test substance in contact with the skin (occlusive barrier).
The adhesive dressings and gauze patches were removed after 24 h contact with the skin and the treated sites gently cleaned by washing with lukewarm water.
- Observations
Approximately 24 and 48 h after removal of the dressings, the treated sites were examined for signs of reaction to treatment.
Scoring scale was according to the following table:
Reaction to treatment Value
No visible change 0
Discrete or patchy erythema 1
Moderate and confluent erythema 2
Intense erythema and swelling 3


Main study - Induction - Intradermal injection
- Allocation
Animals were allocated to treatment to give a test group of 10 animals and a control group of 5 animals.
- Preparation of the site (Day 1)
On the day of dosing (Day 1) the hair over the scapulae was removed over an area of approximately 20x40 mm using an electric clipper with a suitable blade.
- Dosage
Three pairs of intradermal injections were made at the prepared skin site of each animal. All injections were made at the edge of the prepared site and the anterior and median injections were positioned close together and distant from the posterior injections. A volume of 0.1 mL was injected at each site.

- Treatment of the test group
Injection site Treatment
Anterior Emulsified Freund’s complete adjuvant
Median 0.5% test substance in the vehicle (corn oil)
Posterior 0.5% test substance in emulsified Freund’s complete adjuvant

- Treatment of the control group
Injection site Treatment
Anterior Emulsified Freund’s complete adjuvant
Median Vehicle (corn oil)
Posterior Vehicle mixed with emulsified Freund’s complete adjuvant

- Dosing method
The animals were administered by an intradermal injection with a graduated syringe on Day 1.
Observations
Skin reaction at the injection sites was assessed approximately 24 h after injection, according to Draize scoring (described above).

Main study - Induction - Topical application
- Preparation of the site and dosing (Day 8)
7 d after injection (Day 8), the hair was removed on the area surrounding the injection sites by means of an electric clipper. A gauze patch measuring 20x40 mm was soaked with 0.4 mL of the test substance at a concentration of 50% in corn oil or vehicle alone according to the group and then placed over the injection sites. The treatment site was covered with a strip of synthetic film and the trunk wrapped with an elastic adhesive bandage to maintain the test substance in contact with the skin (occlusive barrier).
After a contact period of 48 h, the dressings were removed and the treated sites gently cleaned by washing with lukewarm water.
- Observations
Reaction to treatment was assessed approximately 24 h after removal of the dressings, according to the following table:
Reaction to treatment Value
No visible change 0
Discrete or patchy erythema 1
Moderate and confluent erythema 2
Intense erythema and swelling 3

Main study - Challenge
- Preparation of the site (Day 22)
On Day 22, 3 weeks after preparing the animals for the first induction phase of the main study, all animals were prepared for challenge by clipping the flanks free of hair to expose areas of approximately 50x50 mm on each flank.
- Dosing (Day 22)
A gauze patch measuring 20x20 mm was soaked with 0.2 mL of the test substance at concentration of 20% in corn oil (or vehicle, corn oil) and then placed over the centre of the right (or left) flank. When both sites of the animal had been treated, they were covered with a strip of synthetic film and the trunk wrapped with an elastic adhesive bandage to maintain the test substance and vehicle in contact with the skin (occlusive barrier). After a contact period of 24 h, the dressings and patches were removed and the treated sites were washed with lukewarm water.
- Observations
The treated sites were closely clipped to remove any hair that may have grown approximately 21 h after the removal of the dressings. Observation of the treated sites was carried out approximately 24 and 48 h after removal of the dressings.

The reaction was scored according to the following table:
Reaction to treatment Value
No visible change 0
Discrete or patchy erythema 1
Moderate and confluent erythema 2
Intense erythema and swelling 3
Positive control substance(s):
yes
Remarks:
α-Hexylcinnamaldehyde tested in separate study

Results and discussion

Positive control results:
Sensitisation reactions were observed in 60% of the animals. These results are considered to be acceptable for a weak sensitiser like the α-Hexylcinnamaldehyde.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20% in corn oil (topical exposure)
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 20% in corn oil (topical exposure). No with. + reactions: 10.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20% in corn oil (topical exposure)
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 20% in corn oil (topical exposure). No with. + reactions: 10.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: control
Dose level:
20% in corn oil (topical exposure)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: control. Dose level: 20% in corn oil (topical exposure). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: control
Dose level:
20% in corn oil (topical exposure)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: control. Dose level: 20% in corn oil (topical exposure). No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Preliminary tolerance test

The preliminary tolerance test to establish a suitable concentration for injection in the main sensitisation test indicated that the test substance at a concentration of 0.5% in corn oil was reasonably tolerated. A concentration of 50% was selected for the main topical induction as it was judged to be tolerated by the animals.

Induction

Moderate to severe erythema (score of 3) was seen at the intradermal injection sites following administration of Freund’s complete adjuvant (test and control group, anterior sites), at sites treated with the Freund’s complete adjuvant mixed with 0.5% of the test substance (test group, posterior sites) and at sites treated with the test substance at a concentration of 0.5% in corn oil (test group, median sites). Well defined erythema (score of 2) was seen at sites treated with the Freund’s complete adjuvant mixed with vehicle (control group, posterior sites). None to very slight erythema (scores of 0 and 1) was noted at median sites treated with the vehicle alone (control group). Moderate/confluent to intense erythema and swelling (scores of 2 and 3) were observed following 48 h of topical exposure with the test substance at a concentration of 50% in corn oil (test group). In addition, fissuring of the treated site was observed in test group animals. No erythema was seen in the control group animals. Based on the above results and those seen in the preliminary phase, the test substance at 20% concentration was selected to be used in the challenge phase.

Challenge

Discrete/patchy to intense erythema and swelling (scores ranging from 1 to 3) were observed in all test group animals at the observation performed 24 and 48 h following 24 h topical exposure period to the test substance at 20% concentration in corn oil. No reaction to the vehicle alone was observed in any animal.

Body weight

Body weight changes were not out of the expected range.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the test substance was considered to be a skin sensitizer.
Executive summary:

A guinea pig maximization test was conducted to evaluate the skin sensitization potential of the test substance according to OECD Guideline 406 and EU Method B.6, in compliance with GLP.Based on the results of a preliminary study, 0.5 and 50% in corn oil were selected as intradermal and dermal induction doses. The concentration used for challenge application was 20% in corn oil. All of the animals of the test group showed skin reactions 24 and 48 h after challenge exposure.No reaction to the vehicle alone was observed in any animal.Under the study conditions, the test substance was considered to be a skin sensitizer (Salvador M, 2015).