Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 Mar - 20 May 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A non-LLNA test is available that was performed to comply with Japanese and other non-EU regulations. In accordance with the REACH Regulation (EC) No. 1907/2006, the data were included to avoid unnecessary testing.

Test material

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

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Kisslegg, Germany
- Age at study initiation: 4 - 6 weeks
- Weight at beginning of pre-test: 290 - 395 g
- Weight at beginning of acclimatisation period: 340 - 424 g
- Housing: Individually in Makrolon type-4 cages with standard soft wood bedding ("Lignocel", Schill AG, Muttenz, Switzerland).
- Diet: Pelleted standard Provimi Kliba 3418 guinea pig breeding and maintenance diet (batch no. 82/08), containing Vitamin C (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: Community tap water from Füllinsdorf, ad libitum
- Indication of any skin lesions: Only animals without any visible signs of illness/skin lesions were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Concentration / amount:
Intradermal: 5%
Epidermal: 15%
Day(s)/duration:
Intradermal: three pairs of injections (Day 1); epidermal: 48 h (Day 8)
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
0.01%
Day(s)/duration:
24 h (Day 22)
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
pre-test: 5 males
main study: 10 males (test group), 5 (control group)
Details on study design:
RANGE FINDING TESTS (PRE-TEST):
A. INTRADERMAL INJECTIONS
Four intradermal injections (0.1 m L/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant (FCA) / physiological saline were made into the shaved neck of one guinea pig. Six days later intradermal injections (0.1 mL/site) were made into the clipped flank of the same guinea pig at concentrations of 5%, 3% and 1% of the test item in olive oil. Dermal reactions were assessed 24 h later. Based on the results, the test item concentration of 5% was selected for intradermal induction in the main study.

B. EPIDERMAL APPLICATION
Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant (FCA) / physiological saline were made into the shaved neck of two guinea pigs. Six days later, four patches of filter paper (3 x 3 cm) were saturated with the test item at 15% (the technically highest possible concentration to be applied sufficiently), 10%, 5% and 1% in olive oil and applied to the clipped and shaved flanks of the same guinea pigs. The volume of test item preparation applied was approximately 0.2 mL for the test item at 5% and 1% and an amount of approximately 0.2 g was applied for the test item concentrations of 15% and 10%. No highest non-irritating concentration could be determined after the epidermal pretest. Therefore, a second pretest was performed with two additional naive guinea pigs, treated in the same way as described above, with the concentrations of 0.5%, 0.1% , 0.05% and 0.01% in olive oil, using an application volume of approximately 0.2 rnL. Based on the results obtained, the concentration selected for induction and challenge in the main study were 15% and 0.01% , respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epidermal)
- Site: Scapular area (approximately 6 x 8 cm)
- Frequency of applications: Day 1 (intradermal) and Day 8 (epidermal)

- Test groups:
Intradermal (3 pairs of injections, 0.1 mL/site):
Injection 1: 1:1 (v/v) mixture of Freund's Complete Adjuvant (FCA) and physiological saline
Injection 2: Test item at 5% in olive oil
Injection 3: Test item at 5% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant (FCA) and physiological saline
Epidermal: One week after the injections, a 2 x 4 cm patch of filter paper was saturated with the test item at 15% in olive oil and placed over the injection sites. 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 h.

- Control group:
Intradermal (3 pairs of injections, 0.1 mL/site):
Injection 1: 1:1 (v/v) mixture of Freund's Complete Adjuvant (FCA) and physiological saline
Injection 2: Olive oil
Injection 3: Olive oil in a 1:1 (v/v) mixture of Freund's Complete Adjuvant (FCA) and physiological saline
Epidermal: As described above with olive oil only, applied at a volume of approximately 0.3 mL.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 22
- Exposure period: 24 h
- Test groups: test item and vehicle
- Control group: test item and vehicle
- Site: left flank (test item), right flank (vehicle), 3 x 3 cm each
- Concentrations: 0.01% in olive oil
- Evaluation (hr after challenge): 24 and 48 h

OTHER:
The test item skin area of the animals used for the epidermal pretest and challenge was depilated approximately 21 h after the patches have been removed, using an approved depilatory cream (VEET Cream, Reckitt & Colman AG, Allschwil, Switzerland). The depilation was performed to clean the stratum corneum from possible staining produced by the test item and to facilitate the reading of the possible skin reaction. The depilatory cream was placed on the patch sites and surrounding areas, and left on for up to 3 - 5 min. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.
Positive control substance(s):
yes
Remarks:
α-1-Hexylcinnamaldehyde

Results and discussion

Positive control results:
The positive control was performed in a separate study (Harlan Laboratories Study C16261, conducted from 8 Oct - 14 Nov 2008). ALPHA-HEXYLCINNAMALDEHYDE was used as positive control substance. The study was performed with 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs (CRL:(HA)BR), Charles River Deutschland GmbH, Kisslegg, Germany). The intradermal induction in the test group was performed in the nuchal region with a 1% dilution of ALPHA-HEXYLCINNAMALDEHYDE in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction was conducted for 48 h under occlusion with the test item at 10% in PEG 300 one week after the intradermal induction and following pretreatment of the test areas with 10% Sodium-Lauryl-Sulfate (SLS) approx. 24 h prior to application of ALPHA-HEXYLCINNAMALDEHYDE. The 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 ALPHA-HEXYLCINNAMALDEHYDE at 3% and 1% in PEG 300 and PEG 300 alone under occlusive dressing. Cutaneous reactions were evaluated approx. 24 and 48 h after removal of the dressing. The test yielded the expected results, thus confirming the sensitivity of the test system used.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
intradermal induction: 0%, challenge: 0.01%
No. with + reactions:
5
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
intradermal induction: 0%, challenge: 0%
No. with + reactions:
4
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
intradermal induction: 5%, challenge: 0.01% in olive oil
No. with + reactions:
7
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
intradermal induction: 5%, challenge: 0%
No. with + reactions:
3
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
intradermal induction: 1% in PEG-300, challenge: 3% in PEG-300
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Harlan study C16261, performed from 08 0ct - 14 Nov 2008
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
intradermal induction: 0%, challenge: 0.01%
No. with + reactions:
5
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
intradermal induction: 0%, challenge: 0%
No. with + reactions:
4
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
intradermal induction: 5%, challenge: 0.01% in olive oil
No. with + reactions:
7
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
intradermal induction: 5%, challenge: 0%
No. with + reactions:
3
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
intradermal induction: 1% in PEG-300, challenge: 3% in PEG-300
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Harlan study C16261, performed from 08 0ct - 14 Nov 2008

Any other information on results incl. tables

RESULTS

Mortality

There were no deaths during the course of the study. Hence, no necropsies were performed.

Clinical Signs

No signs of systemic toxicity were observed  in the animals.

Body Weights

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

Skin Effect in the Intradermal Induction

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

Skin Effects in the Epidermal Induction

Control Group: No erythematous or oedematous reaction was observed in the animals treated with olive oil only.

Test Group: As the test item at 15% stained the skin black, it was not possible to determine whether erythema was present or not. However, no oedema was observed.

Skin Effects in the Challenge

Control Group: Discrete / patchy to moderate / confluent erythema was observed in 4 out of 5 control animals at the 24 and 48 h reading when treated with olive oil only. Discrete / patchy to moderate / confluent erythema was observed in all control animals at the 24 and 48 h reading when treated with the test item at 0.01% in olive oil.

Test Group: Discrete / patchy erythema was observed in 3 animals at the 24 and 48 h reading when treated with olive oil only. Discrete / patchy to moderate / confluent erythema was observed in 7 out of 10 test animals at the 24 and 48 h reading when treated with the test item at 0.01% in olive oil.

DISCUSSION AND CONCLUSION

In the challenge, 70% of the test animals showed discrete / patchy erythema (30%) to confluent / moderate erytherna (40%) 24 h after the challenge treatment with the test item at 0.01% in olive oil. With the same treatment, all 5 control animals showed discrete / patchy erythema (40%) to moderate /confluent erythema (60%) at 24 h. At the 48 h reading, the findings persisted in both groups at the same incidence, the same severity in the control group and with slightly less severity in the test group. Since the local reactions in the control group must be due to irritation, they cast doubt on the nature of the reactions observed in the test animals, particularly since they are of the same grade. In such a case one approach is to consider as evidence for sensitization only the skin reactions in the test group animals, which are of a higher grade than the maximum effects observed in the control group animals. However, in the test group, no higher grade than 2 was observed and this was also the maximum score observed in the control group. Additionally, by comparing  the severity  index of each  group, the values obtained in the test group were below the values of the control group, with a range of 0.9 - 1.1 in the test group versus 1.6 in the control group. The severity of the dermal reaction resulting from the challenge application was less marked in the test group when compared to animals of the control group. Moreover, the response decreased in 2 out of 10 test animals at the 48 h reading in the test group. This pattern indicates that the effects detected are not test item related.

As the skin reactions observed excluded an allergenic potential of the test item, the vehicle olive oil had to be considered. 80% of the control animals were affected with a grade 1 or 2 and a severity index of 1 and 30% of the test animals with a grade 1 and a severity index of 0.3 when treated with olive oil alone. Olive oil is one of the best natural conditioners for skin. Its components are readily and quickly absorbed. An excellent moisturiser, it is also a natural delivery system suitable for immune tests, in which the penetration is considered crucial. Evidence exists of the ability of protein and oil components of olive oil to trigger immediate hypersensitivity via IgE antibody and delayed hypersensitivity via cell-mediated immune responses, respectively. This immediate and delayed hypersensitivity of olive oil, though rare, cannot be excluded in the discussion of the effects observed as the sites treated with olive oil alone through the challenges showed local findings. The treatment of the control group with olive oil during the induction phase seemed to enhance the challenge reactions to the test item. This effect is well-known after a pre-treatment of the abdomen of guinea pigs with croton oil, which greatly enhances the challenge reaction to potential allergens. Based on the findings, the challenge reactions failed to appreciate the olive oil's significance as a confounding factor in this procedure. Despite the choice of vehicle, the difference observed in the control and test group with higher severity in the control group and the fading of the skin reaction in a few number of test animals at the 48 h reading suggest that the test item is not sensitizing when applied in combination with a vehicle well-known for its permeation enhancement.

INDIVIDUAL TABLES

Table 1: Skin reactions in the intradermal pretest

Animal no.

Concentration test item in olive oil [%]

Reaction after 24 h

1452

5

2

3

2

1

1

 

Table 2: Skin reactions in the epidermal pretest

Animal no.

Concentration test item in olive oil [%]

Reaction after removal of bandage

24 h

48 h

1453

15

1

1

10

1

1

5

1

1

1

1

1

1454

1

1

1

15

1

1

10

1

1

5

1

1

1470

0.5

1

1

0.1

1

1

0.05

1

1

0.01

0

0

1471

0.01

0

0

0.5

1

1

0.1

1

1

0.05

1

0

 

Skin reactions after challenge

Table 3: Control group treated with olive oil

Animal no.

Reaction after removal of bandage

24 h

48 h

1455

0

0

1456

1

1

1457

1

1

1458

1

1

1459

2

2

 

Table 4: Control group treated with test item (0.01% in olive oil)

Animal no.

Reaction after removal of bandage

24 h

48 h

1455

1

1

1456

2

2

1457

1

1

1458

2

2

1459

2

2

 

Table 5: Test group treated with olive oil

Animal no.

Reaction after removal of bandage

24 h

48 h

1460

0

0

1461

0

0

1462

0

0

1463

0

0

1464

0

0

1465

0

0

1466

1

1

1467

1

1

1468

1

1

1469

0

0

 

Table 6: Test group treated with test item (0.01% in olive oil)

Animal no.

Reaction after removal of bandage

24 h

48 h

1460

2

1

1461

2

1

1462

1

1

1463

0

0

1464

1

1

1465

2

2

1466

1

1

1467

2

2

1468

0

0

1469

0

0

 

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008
Conclusions:
Based on the findings of the present adjuvant sensitization test in guinea pigs, the test item is not considered to be a skin sensitizer.