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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
guinea pig maximisation test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 February - 25 May 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with International guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

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
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study pre-dates the adoption of the OECD 429 OECD Guideline (2002). The most appropriate method at the time was conducted for this study.

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol
Cas Number:
1478-61-1
Molecular formula:
C15H10F6O2
IUPAC Name:
4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]diphenol
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, dark.
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Not reported
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item formulated in corn oil within fours prior to treatment.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid (formulated in corn oil)

OTHER SPECIFICS: Homogeneity of the formulation was confirmed by visual inspection.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Not reported
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: 378 - 467 g
- Housing: Grouped housing of 5 animals per metal cage with wire-mesh floors.
- Diet (e.g. ad libitum): Free access to standard guinea pig diet, including ascorbic acid (1000 mg/kg) (Charles River Breeding and Maintenance Diet for Guinea Pigs, Altorim, Germany)
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: Minimum of 5 days
- Indication of any skin lesions: Not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12:12 light:dark
- IN-LIFE DATES: From: To: Not reported

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Intradermal injection: 1 %
Epidermal: 50 %
Day(s)/duration:
Epidermal: 48 h contact
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
5 % concentration (0.1 mL)
Day(s)/duration:
24 h contact
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Treated: 10
Control: 5
Details on study design:
RANGE FINDING TESTS:

Formulations of the test item were prepared in corn oil.

Intradermal test- A series of 6 test item concentrations were used; 20, 10, 5, 2, 1 and 0.5 % (top concetration being the highest that could technically be injected) with each of the three animals recieving two injections (0.1 mL) each i.e. 20 and 10 % tested in one animal, 5 and 2 % in the second etc. 1 % was found to cause moderate irritation and was chosed as the concentrtion to be used for the induction phase of the main test.

Epidermal test- Four concentrations were used; 50, 20, 10 and 5 % (top concentration being the highest that could be technically applied) with each of the four animals treated with two concentrations each i.e. test concentrations were tested in duplicate. 0.5 mL solution wa applied to clipped flanks and covered for 24 h. Residual test item was then removed and the skin cleaned using water. Observations were taken after 24 and 48 h contact time. The 5 % solution would be used in the challenge phase of the main test as it was the highest concetration that did not cause irritation. The 50 % solution would be used in the main test as it was the maximum concentration found to cause moderate irritation.

MAIN STUDY
A. INDUCTION EXPOSURE - Intradermal injection
- No. of exposures: 1
- Exposure period: n/a
- Test groups: 10 animals each injected at 3 sites (injections in pairs either side of the midline) (1. mixture of Freunds' Complete Adjuvant (FCA) and water, 2. test item at 1 % concentration , 3. test item and FCA 1:1 v/v (1 % concentration of test item)
- Control group: 5 animals
- Site: Scapular region
- Frequency of applications: Once
- Duration: n/a
- Concentrations: 1 %

A. INDUCTION EXPOSURE - Epidermal injection
- No. of exposures: 1
- Exposure period: 48 h
- Test groups: 10 animals
- Control group: 5 animals
- Site: scapular region, between injection sites
- Frequency of applications: Once
- Duration: 14 days
- Concentrations: 1 %

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 21
- Exposure period: 24 h
- Test groups: 10 animals
- Control group: 5 animals
- Site: flank
- Concentrations: 5 %
- Evaluation (hr after challenge): 24 and 48 h after application

OTHER:
Challenge controls:
Corn oil
Positive control substance(s):
yes
Remarks:
Assessment study conducted within last 6 months successfully validated procedure. Reference item was alpha-hexylcinnamicaldehyde.

Results and discussion

Positive control results:
100 % sensitisation observed in test group

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10 % (alpha-hexylcinnamicaldehyde)
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
NONE
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10 % (alpha-hexylcinnamicaldehyde)
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
None
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Table 1:       Erythema gradings after induction and challenge phases

Rep.

Intradermal

Epidermal

3 days after induction

10 days after induction

23 days after induction (1 day after challenge)

24 days after induction (2 days after challenge)

A

(Erythema Score)

B

(Erythema Score)

C

(Erythema Score)

Erythema Score

Oedema Score

5 %

Control

5 %

Control

Control

1

3

2

2

2

0

0

0

0

0

2

3

2

3

0

0

0

0

0

0

3

3

3

3

0

0

0

0

0

0

4

3

2

3

2

0

0

0

0

0

5

3

1

3

2

0

0

0

0

0

Treatment

1

2

1

2

3

0

0

0

0

0

2

3

3

3

3

0

0

0

0

0

3

3

3

3

3

0

0

0

0

0

4

3

2

3

3

0

0

0

0

0

5

3

2

3

3

0

0

0

0

0

6

2

2

2

n

1

0

0

0

0

7

3

2

3

3

0

0

0

0

0

8

3

2

3

3

0

0

0

0

0

9

3

2

2

3

0

0

0

0

0

10

3

3

3

n

1

0

0

0

0

A: 1:1 mixture of FCA and water for injection

B: Corn oil (control) or 1 % test item (treatment)

C: Corn oil (control) or 1:1 mixture of FCA and test tem (1 % final concentration) (treatment)

Epidermal induction: Corn oil (control) or 50 % test item (treatment)

Epidermal challenge: Corn oil (control) or 5 % test item (treatment)

n: necrosis observed

Grading’s on a scale of 0 – 4 (0: no erythema / no oedema, 1: slight erythema / slight oedema, 2: well-defined erythema, 3: moderate erythema and swelling, 4: severe erythema, swelling and deep epidermal injury)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item cannot be classified as a skin sensitiser based on GHS criteria.
Executive summary:

In a dermal sensitization study (OECD 406) with test item in corn oil, young adult Dunkin Hartley albino guinea pigs (15 females) were tested using the guinea pig maximisation method.

A preliminary irritation study was conducted using 3 individuals in order to select the test substance concentrations to be used in the main study. The results concluded that intradermal injections at a concentration of 1 % test item and epidermal application at a concentration of 50 % test item were the highest concentrations that produced moderate irritation. These concentrations were therefore assigned in the induction phase of the main test. The highest epidermal concetration applied that did not cause irritation was 5 % test item. This concetration was therefore assigned in the challenge phase of the main test.

The main test was conducted using 15 individuals, 5 controls and 10 treated. On Day 1, each individual was subjected to three pairs of intradermal injection around the scapular region (0.1 mL/ site); 2 x 1:1 FCA:water, 2 x 1 % test item in corn oil (or corn oil only for controls), 2 x 1 % test item in FCA (1:1 mix) (corn oil: FCA mix for controls). The dermal reactions cused by the injections were assessed on Day 3 for irritation. On Day 8, the scapular region was clipped and a dermal application (0.5 mL) of the test item (50 % concentration) was applied (2 x 3 cm patch) between the injection sites. The patch was held in place for 48 using micropore tape and an elastic bandage. The patches were removed and the applications sites cleaned prior to assessment. On Day 21 each individual recieved a challenge dermal application, that was applied as previously described, at a concetration of 5 %. The patch was removed after 24 h contact time, the skin cleaned and assessment for irritation made at 24 and 48 h (after challenge application).

The results of the main studies showed moderate erythema (grade 3) at the injection and dermal patch sites after the induction of the test item at a concentration of 1 % and 50 %, respectively. It should be noted that signs of necrosis was observed in 2 treated individuals after the dermal induction phase. No skin reactions were observed following the challenge phase in any of the individuals. No mortality or systemic toxicity were observed during the main test and body weight increases in the treated individuals was in the same range as the controls.

In this study, the test item is not a dermal sensitizer.