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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:
12 Mar - 14 Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted in 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Slovenska Narodna Akreditacna Sluzba, Bratislava, Slovenska Republika
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
As the test substance is likely to have surfactant properties, the default LLNA study (OECD 429) is not recommended in this case.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
albino
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Velaz, Czech Republic
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 10 weeks old
- Weight at study initiation: 462 – 517 g
- Housing: up to 5 animals per cage in plastic cages suspended on stainless steel racks with Lignocel S3/4 bedding (Lufa-ITL GmbH, Germany)
- Diet: laboratory food ssniff (Spezialdiäten GmbH, Germany), offered in recommended doses each day approximately at the same time (analyses were performed)
- Water: tap water (containing 0.05% ascorbic acid), ad libitum (analyses were performed)
- Acclimation period: 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 0.3
- Humidity (%): 58.8 ± 2.2
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: ethanol
Concentration / amount:
2%
Day(s)/duration:
Day 0 / single injection
Adequacy of induction:
other: highest concentration that did not cause destructive concentration of the skin after 24 h
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
10%
Day(s)/duration:
Day 7 / 48 h
Adequacy of induction:
other: minimal irritating concentration
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol
Concentration / amount:
2%
Day(s)/duration:
Day 21 (treated group) and 22 (control group) / 24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Range finding test: 3 females

Main test:
Test group: 20 females
Negative control group: 10 females

Positive control group: 5 females
Details on study design:
RANGE FINDING TESTS:
Three females were pre-treated intradermally with an injection of 0.1 mL of FCA mixed 1:1 with vehicle (ethanol) 24 h before the induction phase.
These animals were used to assess the primary irritation tests after intradermal injection and after topical application. One animal was treated intradermally with 6 different test substance concentrations (initially 10, 25 and 50% on the right side of the animal; on the next day 1, 2 and 8% on the left side of the animal) and two animals were treated topically (4 different test substance concentrations each: 25, 50, 75 and 100% on one animal and the next day 2, 5, 10 and 15% on the second animal). Observations of the skin for the primary irritancy were scored after 24 h according to the Draize et al. scale. At a test substance concentration of 1%, the intradermal injection caused very slight erythema (grade 1) and no edema, well-defined erythema (grade 2) and very slight oedema (grade 1) were observed at concentrations of 2% or more. After topical application no edema was observed at any concentration, while very slight erythema (grade 1) was observed at 5% and well-defined erythema (grade 2) at 10% or more. According to the results of the range finding study, the following dose levels were selected for the main study:
Induction / intradermal injection: 2%
Induction / epicutaneous application: 10%
Challenge / epicutaneous application: 2%

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

- Test groups:
Intradermal (three pairs of injections; 0.1 mL/site):
Injection 1: Freund's Complete Adjuvant (FCA) 1:1 with water
Injection 2: 2% test substance formulated in vehicle
Injection 3: 2% test substance formulated in a 1:1 mixture FCA/water

Epicutaneous:
The test substance (in the minimal irritating concentration, 10%) was spread over 2 x 4 cm piece of gauze to saturation. The patch was secured with an occlusive dressing for 48 h.

- Control group:
Intradermal (three pairs of injections, 0.1 mL/site):
Injection 1: FCA 1:1 with water
Injection 2: undiluted vehicle
Injection 3: 50% w/v formulation of the vehicle in a 1:1 mixture FCA/water

Epicutaneous:
The vehicle was applied in the same manner as the test item in the treated group and held in contact by an occlusive dressing for 48 h.

- Site: shoulder region
- Frequency of applications: once (intradermal induction on Day 0 and epicutaneous application on Day 7)
- Concentrations: 2% (intradermal) and 10% (epicutaneous)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 21 (treated group), 22 (control group)
- Exposure period: 24 h
- Test groups: test substance and vehicle
- Control group: test substance and vehicle
- Site: flanks, previously unexposed area
- Concentrations: 2% of test substance (epicutaneous application)
- Evaluation: 24 and 48 h after removing the patches

OTHER:
The animals were inspected daily during the study for clinical symptoms. Individual weights of animals were determined shortly before the test substance was applied and at the end of the study.
Challenge controls:
the control group is actually a challenge control
Positive control substance(s):
yes
Remarks:
α-hexylcinnamaldehyde (in concurrent study, reliability check)

Results and discussion

Positive control results:
The reliability of experimental technique used was performed with five animals, using α-hexylcinnamaldehyde (positive control) as a part of the study.
The same procedure as on treated and control animals were used. A pilot study was not performed due to the fact that the used substance (α-hexylcinnamaldehyde) is well known. The applied concentrations were chosen based on literature and previous studies.
Induction / Intradermal injection: 0.3%
Induction / epicutaneous application: 100% (undiluted)
Challenge / epicutaneous application: 100% (undiluted)
Well defined erythema (grade 2) was seen 24 h after removal of the patches in 2/5 animals of the positive control group, and slight erythema (grade 1) for the remaining 3/5 animals. This result represents skin sensitization reaction in 100% of tested animals. Slight erythema (grade 1) was still visible 48 h after removal of the patches in 4/5 animals. Edema was not observed in any of the animals. Thus the reliability of the experimental technique could be shown.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
intradermal induction: 0%; challenge: 2%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
intradermal induction: 2% challenge: 2%
No. with + reactions:
5
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
intradermal induction: 0.3%, challenge: 100%
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
intradermal induction: 0% challenge: 2%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
intradermal induction: 2% challenge: 2%
No. with + reactions:
2
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
intradermal induction: 0.3%, challenge: 100%
No. with + reactions:
4
Total no. in group:
5
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Table 3: Skin reaction in Range finding study

Animal No.

Test substance concentration (%)

Erythema

Edema

Route

1

50

2

1

intradermal
injection

25

2

1

10

2

1

8

2

1

2

2

1

1

1

0

2

100

2

0

epicutaneous application

75

2

0

50

2

0

25

2

0

3

15

2

0

epicutaneous application

10

2

0

5

1

0

2

0

0

Table 4: Skin response after challenge (treated with 2% of the test substance) and body weight change

Animal No.

Group

Reaction scores

Body weight change/ gain (g)**

24 h*

48 h*

Erythema

Edema

Erythema

Edema

1

Control group

0

0

0

0

36

2

0

0

0

0

52

3

0

0

0

0

36

4

0

0

0

0

86

5

0

0

0

0

54

6

0

0

0

0

46

7

0

0

0

0

62

8

0

0

0

0

76

9

0

0

0

0

90

10

0

0

0

0

48

11

Treatment group

0

0

0

0

66

12

0

0

0

0

38

13

0

0

0

0

30

14

0

0

0

0

64

15

0

0

0

0

74

16

0

0

0

0

68

17

1

0

1

0

38

18

0

0

0

0

72

19

0

0

0

0

10

20

0

0

0

0

62

21

1

0

0

0

70

22

1

0

0

0

46

23

0

0

0

0

38

24

1

0

1

0

32

25

0

0

0

0

56

26

0

0

0

0

54

27

0

0

0

0

70

28

1

0

0

0

84

29

0

0

0

0

6

30

0

0

0

0

42

* = time after removal of the patches

** =  before test substance application and at end of the study

Table 5: Skin response and body weight change after treatment with undiluted α-hexylcinnamaldehyde (positive control)

Animal No.

Reaction scores

Body weight change/ gain (g)**

24 h*

48 h*

Erythema

Edema

Erythema

Edema

1

1

0

1

0

18

2

1

0

0

0

48

3

2

0

1

0

48

4

1

0

1

0

60

5

2

0

1

0

32

* = time after removal of the patches

** = before test substance application and at end of the study

No clinical signs and no deaths in any of the groups were observed. The body weights of all animals were increasing during the study.

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:
In this guinea pig maximisation test (GPMT) 25% of the animals revealed a positive response after challenge at an intradermal induction concentration of 2%. Thus, the substance is not considered a skin sensitiser according to Regulation (EC) No. 1272/2008.