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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From July 17 to 24, 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Complete read-across justification is attached in section 13. Source study has reliability 1.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1996
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Guinea pig maximisation test was available.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Himalayan spotted
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Biotechnoligy & animal breeding division, Wölferstrasse 4, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: 328 - 347 g
- Housing:individually in Makrolon type-4 cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 week for the control and test group under test conditions after health examination. No acclimatisation for the animals of the pretest.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 22.5 °C
- Humidity: 45 - 73 %
- Air changes: 10 - 15 per h
- Photoperiod: 12 h light and 12 h dark

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
polyethylene glycol
Concentration / amount:
10 %
Day(s)/duration:
on day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal
Vehicle:
physiological saline
Remarks:
and Freund's complete adjuvant 1:1 v/v
Concentration / amount:
10 %
Day(s)/duration:
on day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
40 %
Day(s)/duration:
on 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:
40 %
Day(s)/duration:
on day 22
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
3 animals in pretest
5 animal in control group
10 animals in test group
Details on study design:
RANGE FINDING TESTS
INTRADERMAL INJECTIONS
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig.
One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of B = 10 % and C = 5 % of the test item in PEG 300.
The concentration of A = 20 % was not injectable. The three concentrations were determined during formulation trials performed prior to the pretest. Although the 20 % test item concentration was prepared, the concentration of 10 % was considered to be the highest technically applicable concentration which could be injected into the intra-cellular space in spite of the high viscosity of the application dilution and the obstacle caused by the tissues.
Dermal reactions were assessed 24 hours later.
Based on the results, the test item concentration of 10 % was selected for intradermal induction in the main study.

EPIDERMAL APPLICATIONS
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (3 cm × 3 cm) were saturated with the test item at D = 40 % (technically the highest possible concentration to be applied sufficiently), E = 20 %, F = 10 % and G = 5 % in PEG 300 and applied to the clipped and shaved flanks.
The amount of test item preparation applied was approximately 0.2 g for the test item at 40 % and a volume of approximately 0.2 ml was applied for the remaining test item concentrations. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test item. The dressings were removed after an exposure period of 24 hours.
24 hours after removal of the dressing the application site was depilated in order to visualize any resulting erythema. The depilatory cream was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. Thereafter, the animals were dried with a disposable towel, and returned to their cages.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.
The allocation of the different test item dilutions to the sites (D, E, F, G) on the two animals was alternated in order to minimize site-to-site variation in responsiveness. Based on results obtained, the concentration selected for induction and challenge in the main study was 40 %.

MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: 1
- Day of injections: day 1
- Test groups: 1
- Control group: 1
- Site: scapular region
- Frequency of applications: 3 pairs of injections
- Concentrations:
test group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test item, at 10 % in PEG 300.
3) The test item at 10 % in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
control group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) PEG 300
3) 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.


A. INDUCTION EXPOSURE: EPIDERMAL
On test day 7 and 23 hours prior to the epidermal application the scapular area of the animals of the control and test group was clipped, shaved free of hair and the test area was pretreated with 0.5 ml of 10 % sodium-lauryl-sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass rod without bandaging. This 10 % concentration of SLS enhances sensitization by provoking a mild inflammatory reaction.
- No. of exposures: 1
- Day of application: day 8
- Exposure period: 48 h
- Test groups: 1
- Control group: 1
- Site: scapular region
- Frequency of applications: 1
- Concentrations: on test day 8, a 2 cm × 4 cm patch of filter paper was saturated with the test item (40 % in PEG 300) and placed over the injection sites of the test animals. The amount of test item preparation applied was approximately 0.3 g. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test item. The guinea pigs of the control group were treated as described above with PEG 300 only, applied at a volume of approximately 0.3 ml.


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure period: 24 h
- Test groups: 1
- Control group: 1
- Site: flank
- Concentrations: 2 patches (3 cm × 3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 40 % (applied to the left flank) and the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The amount of test item preparation applied was approximately 0.2 g and a volume of approximately 0.2 ml was used for the vehicle.
- Evaluation (hr after challenge): 24 and 48 h after removal of bandage
Challenge controls:
PEG 300 applied to right flank
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
40 % in PEG
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
40 % in PEG
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Skin effects after intradermal induction performed on test day 1

The expected and common findings were observed in the control and test group after the different applications using FCA intradermally. These findings consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

Skin effects after epidermal induction performed on test day 8

Control group: discrete/patchy erythema was observed at the 24- and 48-hour reading in four out of 5 animals after treatment with PEG 300 only.

Test group: discrete/patchy erythema was observed in all animals at the 24- and 48-hour reading after treatment with the test item at 40 % in PEG 300. Although the test item at 40 % stained the skin yellow, it was still possible to determine whether erythema was present or not. The reactions observed in both groups occurred following pretreatment with 10 % SLS in paraffinum perliquidum.

Skin effects after challenge - performed on test day 22

Control group: no skin reactions were observed in the animals when treated with either PEG 300 only or when treated with the test item at 40 % in PEG 300. Yellow discoloration produced by the test item was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 hours prior to challenge reading.

Test group: no skin reactions were observed in the 9 surviving animals when treated with either PEG 300 only or when treated with the test item at 40 % in PEG 300. Yellow discoloration produced by the test item was noted directly after removal of the patch. To remove the discoloration all animals were depilated 3 hours prior to challenge reading.

Viability / mortality / macroscopic findings: one animal of the test group was found dead on test day 18 (i.e., 6 days after the 48-hour reading of the epidermal induction phase). At necropsy, congestion in the lungs was noted. The cause of death could not be established.

Clinical signs, systemic: no signs of systemic toxicity were observed in the animals.

Body weights: body weight of the animals was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to the CLP Regulation (EC 1272/2008)
Conclusions:
Not skin sensitising in a guinea pig maximisation test.
Executive summary:

Method

A maximisation test was performed in 15 (10 test and 5 control) male albino guinea pigs to assess the skin sensitising potential, in accordance with OECD guideline 406 and EU method B.6. The intradermal induction of sensitisation in the test group was performed in the nuchal region with a 10 % dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline.

The epidermal induction of sensitisation was conducted for 48 hours under occlusion with the test item at 40 % in PEG 300 one week after the intradermal induction and following pretreatment of the test areas with 10 % Sodium- Lauryl-Sulfate (SLS) approximately 23 hours prior to application of the test item.

Animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion following pretreatment with 10 % SLS.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 40 % in PEG 300 and PEG 300 alone under occlusive dressing for 24 hours.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

Results

Skin reactions after the challenge procedure

control group after 24 h after 48 h

% of positive 

(in 5 animals)

test substance 40 % in PEG 300 0 0
PEG 300 only 0 0
test group

% positive

(in 9 animals)*

test substance 40 % in PEG 300 0 0
PEG 300 only 0 0

* One animal was found dead on test day 18. At necropsy, congestion in the lungs was noted. The cause of death could not be established.

No signs of toxicity were evident in the guinea pigs of the control or test group. None of the 9 surviving test animals showed skin reactions after the challenge treatment at 40 % (w/w) in PEG 300. No skin effect was observed in the control group. The substance was considered as not skin sensitising.