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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Feb 2007 - 09 Mar 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
A study was performed according to GLP and the methods applied are fully compliant with OECD TG 406.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
July 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
September 30, 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Guinea pig maximisation test was performed before the LLNA was set as preferred test method.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: HsdPoc:DH
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelman GmbH, Borchen
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: about 3 - 5 weeks
- Weight at study initiation: mean 325 g (range from 271 to 376 g). The body weight of two animals at the start of the study was outside the range as described in the study plan (300 to 450 g)
- Housing: Five guinea pigs were housed in type GMI5 (EBECO) Makrolon cages with a shelter and placed on mobile racks. The animals were kept on conventional softwood granulate as the bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 to 23 °C
- Humidity (%): 55 to 62%

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
paraffin oil
Concentration / amount:
25 g/L with and without Freund's complete adjuvant
Day(s)/duration:
intradermal injection on experimental day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
7.5 g/L
Day(s)/duration:
for 48 hours on experimental day 8
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
2.5 g/L
Day(s)/duration:
for 24 hours, 2 weeks after topical induction
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Total: 20 females
Pre-test: 5 females
Control group: 5 females
Test group: 10 females
Details on study design:
RANGE FINDING TESTS:
To determine the suitable concentrations for the main study, a pretest with intradermal or topical administrations of the vehicles and of the test item preparations were performed. 6 intradermal (i.d.) injections with different concentrations were given to one animal. Two further animals instead of four animals were used to determine the topical concentrations for the main study. One of the pretest animals died on day 6 of the experimental part and one pretest animal was killed in a moribund state on day 15. Pathology investigation in these two animals revealed severe emaciation. In addition, a beginning bridging of teeth in the lower jaw was observed. The one animal which died on day 6 exhibited additionally a pyogranulomatous, partly necrotizing bronchopneumonia with giant cells and some plant material which is indicative for an aspiration pneumonia. Probably as a result of this aspiration pneumonia the soiled fur observed in the neck, breast and foreleg region resulted. Histopathology of this skin region showed a mild neutrophilic dermatitis with intracorneal pustules and orthokeratotic hyperkeratosis. All other histopathology findings in this animal are incidental or a result of the poor animal condition (f.e. lymphoid depletion in the spleen). No histopathology was performed for the animal which was sacrificed on day 15. Overall the death and moribund condition of these two animals is probably the result of poor breeding conditions (beginning bridging of teeth, emaciation, aspiration pneumonia) and not related to the intradermal administration of the test item.
The other two guinea pigs were exposed for 48 hours without pretreatment to 4 different concentrations to determine the slightly irritating concentration for the dermal induction. In the pretest the following concentrations were used:

1. Test material with liquid paraffin as vehicle:
no pretreatment: intradermal injection: 50, 25, 10, 5, 1, and 0 g/L
2. Test material with polyethylene glycol 400 as vehicle:
no pretreatment: topical exposure (48 hours): 100, 10, 7.5, 5, 2.5, and 1 g/L

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (injection and topical application)
- Exposure period: 48 h (topical application)

Group 1 (vehicle: Liquid paraffin)
- cranial: 0.1 mL Freund's complete adjuvant + sodium chloride solution
- medial: 0.1mL Liquid paraffin
- caudal: 0.1 mL Freund's complete adjuvant + sodium chloride solution

Group 2 (test material)
- cranial: 0.1 mL Freund's complete adjuvant + sodium chloride solution
- medial: 0.1 mL test material (25 g/L ad liquid paraffin)
- caudal: 0.1 mL Freund's complete adjuvant with test material
+ sodium chloride solution (25 g test material/L preparation)

- Site: each side of the spinal column in a total area of 2 x 4 cm
1. injection induction: shoulder region (cranial, medial, caudal)
2. topical induction: shoulder region
- Frequency of applications: weekly
- Duration: 48h (topical induction)
- Concentrations: 0.1 mL (intradermal), 0.1 mL (topical application) with group1: polyethylene glycol, group 2: test material (7.5 g/L in polyethylene glycol)

B. CHALLENGE EXPOSURE
Two weeks after topical induction the challenge was performed by fixing two filter papers of about 4 cm2, one fully loaded with 0.5 ml of the test material preparation and soaked with 0.5 ml polyethylene glycol 400, to the shaven side of the animals.
- No. of exposures: 2
- Day(s) of challenge: 14 (challenge 1), 28 (challenge 2)
- Exposure period: 24 h
- Test groups:
Group 1:
- Topical induction: Polyethylene glycol 400 (undiluted)
- Challenge: Polyethylene glycol 400 (undiluted)
- Primary irritation: Test material (2.5 g/L ad polyethylene glycol 400)

Group 2:
- Topical induction: Test material (7.5 g/L ad polyethylene glycol 400)
- Challenge: Test material (2.5 g/L ad polyethylene glycol 400)
- Challenge: Polyethylene glycol 400 (undiluted)
- Site: flank
- Evaluation (hr after challenge): 48 and 72 h after start of challenge

OTHER:
MAIN STUDY-METHOD

+ ++ +++
/__________________/__________________/__________________/_______/
1 8 22 25 day of experimantal part

induction: stage I + = intradermal injection
stage II ++ = topical application
challange:+++ = topical challange

Induction was performed in two stages. First, the test material was intradermally injected with or without Freund's complete adjuvant on day 1. Then, after one week (day 8), the test material was topically applied under occlusive patch conditions for 48 hours.
The challenge was performed two weeks after topical induction by topical application at a non irritating concentration under the same conditions. The patches were then applied for 24 hours on the shaven side of the animals.

-- Observations
During the induction phase, the guinea pigs were examined on days 2, 3, 7, 11,12,15, and 22 of the experimental part for local skin reactions and the results were documented.
The behavior and general condition of all animals were monitored daily.
The challenge sites were investigated for reactions 48 hours after start of the challenge. Further inspections followed 72 hours after start of the challenge, to detect weak or slowly developing reactions.

After the challenge, skin changes at the application sites were evaluated according to the following MAGNUSSON and KLIGMAN scheme.
Reaction type scores Definition
Negative reaction 0 no reaction
positive reaction 1 scattered mild redness
2 moderate and diffuse redness
3 intense redness and swelling
Following the grading according to the EEC Directive 2001/59/EEC, a result in an adjuvant method is considered positive if 30 % or more of the animals showed positive reactions.

Body weight
The body weight of the guinea pigs was determined prior to the start of the experimental part (day 1), on experimental days 8, 15, 22, and at the end of the experimental part.
Challenge controls:
yes (vehicle only)
Positive control substance(s):
yes
Remarks:
Hexylcinnamaldehyde 10 g/L, study T16507

Results and discussion

Positive control results:
50% positive reactions with control substance

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
25 g/L (induction I), 7.5 g/L (induction II), 2.5 g/L (challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
25 g/L (induction I), 7.5 g/L (induction II), 2.5 g/L (challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
PEG400 undiluted
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
PEG400 undiluted
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
10 g/L alpha-Hexylcinnamaldehyde
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
10 g/L alpha-Hexylcinnamaldehyde
No. with + reactions:
5
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Pretest data

For the main study the following concentrations were chosen:

Vehicle:       Liquid paraffin (for intradermal induction)

Polyethylene glycol 400 (for topical induction and challenge)

Test material

Intradermal induction:        25 g/L slightly irritant

Topical induction:               7.5 g/L slightly irritant (48 hours exposure)

Topical challenge:              2.5 g/L not irritant (24 hours exposure)

Main test:

Findings in the induction phase

After intradermal injection, the common signs of irritation after injection of Freund's complete adjuvant were observed. The injection sites were swollen and red, later on, scabs developed.

Findings after challenge

Group 1: negative control group

After challenge with polyethylene glycol 400 (PEG400), no erythema or edema were observed at the readings.

A single treatment was performed with the test material (2.5 g/L PEG400) to exclude the primary irritation potential of the test material. No positive reactions were observed in the treated areas at any reading.

Group 2: test material group

The challenge was performed on the right flank with the test material preparation in PEG400.

No positive skin reactions were observed in the areas treated with PEG400 alone at any reading.

After challenge with the test material (2.5 g/L preparation in PEG400) no positive skin reactions were observed at the readings. This results in 0 % positive reactions at challenge.

Clinical findings and mortality

The clinical behavior of the guinea pigs was normal during the experimental part. All animals survived the experimental part.

Body weight

The body weight development corresponded to that of the animals of the vehicle group.

Applicant's summary and conclusion

Interpretation of results:
other: EU GHS criteria not met
Conclusions:
Under the given experimental conditions, the test material induced no reactions. According to Regulation (EC) No 1272/2008, the test material is not classified as a skin sensitiser.
Executive summary:

The registered substance was tested for skin sensitisation in a guinea pig maximisation test according to OECD Guideline 406 following GLP.

Purpose

The purpose of this GPMT assay was to identify the contact allergenic potential of the test material. This study should provide a rational basis for risk assessment to the sensitising potential of the test item in man.

Study Design

The test material was investigated for skin sensitising properties in the guinea pig maximization test according to MAGNUSSON and KLIGMAN (1969).

5 females were treated with the vehicles liquid paraffin and polyethylene glycol 400 (group 1) and 10 females were treated with the test material (group 2).

Induction included intradermal injection of test material preparation in liquid paraffin (25 g/L with and without Freund's complete adjuvant) on experimental day 1, and topical application of test material preparation in polyethylene glycol 400 (7.5 g/L) for 48 hours on experimental day 8.

Challenge by topical application of the test material preparation in polyethylene glycol 400 (2.5 g/L) for 24 hours was performed two weeks after topical induction and readings were taken at 48 hours and 72 hours after start of treatment.

Results

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. After challenge no positive reactions were seen 48 or 72 hours after treatment with the test material.

Conclusion

Under the given experimental conditions, the test material induced no reactions. According to Regulation (EC) No 1272/2008, the test material is not classified as a skin sensitiser.