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Administrative data

Description of key information

Skin Sensitisation

Under the conditions of the study, the test material is not considered to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 September 2006 to 20 December 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiences which do not affect the quality of the relevant results.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study coducted for worldwide regulatory purposes.
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 503226
- Expiration date of the lot/batch: March 01 2008

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in well closed containers (21 to 25 °C)
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 to 7 weeks old
- Weight at study initiation: 334 to 394 g
- Housing: animals were housed individually or in groups of 2 animals in stainless-steel wire-mesh cages (w 266 x d 266 x h 200 mm)
- Diet:pellet diet (ad libitum)
- Water: Tap water was supplied via an automatic water supplying system.
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 23 ± 5 °C (measured values: 19 to 28 °C)
- Humidity: 55 ± 25 % (measured values: 36 to 85 %)
- Air changes: 11 to 14 times per hour
- Photoperiod: 12-hour lighting (07:00 to 19:00)

Route:
other: intradermal and topical
Vehicle:
other: liquid paraffin for intradermal and polyethylene glycol 300 for topical induction and challenge
Concentration / amount:
25 % (w/w) for intradermal, 50 % (w/w) for topical.
Day(s)/duration:
Intradermal induction on day 1, Topical induction on day 6 (patch applied for 48 hours)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
other: Topical
Vehicle:
polyethylene glycol
Remarks:
300
Concentration / amount:
50 % (w/w) and 25 % (w/w)
Day(s)/duration:
Twenty-one days after the first intradermal injection the challenge was applied for 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 animals for the test material group and 5 animals for the control group.
Details on study design:
PRELIMINARY STUDY TO SELECT DOSE CONCENTRATIONS
-Two selected animals were clipped on their dorsal skin and 0.1 mL of test material suspension (at 25, 10, 5 and 1 % w/w) was injected intradermally to sites A to D.
-Two animals were then clipped and shaved on the scapula area and 0.1 mL of 1:1 emulsion of physiological saline and Freund's complete adjuvant (FCA) was injected intradermally to the four corners of the area. Seven days later fur of 3 x 7 cm on the right and left flanks was clipped and shaved and 0.1 mL of each test material suspension (50, 25, 10, 5, 2.5 and 1 % w/w) was applied to a patch 1.5 cm in diameter. The patch was applied to the shaved skin and covered occlusively with a polyethylene film tape. The patch was removed after 24 hours and the site cleaned with absorbent cotton soaked with polyethylene glycol 300.
-Animals were observed for skin reactions at 24 and 48 hours after intradermal injection or patch removal.

MAIN STUDY

A. INDUCTION EXPOSURE
-Site: On the day before intradermal induction, fur of a 4 x 6 cm area above the scapula was clipped with an electric clipper and further shaved with an electric shaver as closely as possible.
- Intradermal induction: On the next day, 0.1 mL of each intradermal sensitiser used in this study was intradermally administered to the induction sites (A, B and C) within the 2 x 4 cm area.
- Topical induction: On day 6 after intradermal induction, the same area was clipped and shaved with an electric clipper and shaver and 0.2 mL of white petroleum containing 10 % SLS was applied (to D (2 x 4 cm)) between the intradermal induction sites. On the next day, this area was cleaned with absorbent cotton soaked with ether, 0.2 mL of each topical sensitiser was applied to a patch 2 x 4 cm and the patch was applied to the induction area (D) and covered occlusively with a polyethylene film tape for topical induction. The patch was removed 48 hours after application.

B. CHALLENGE EXPOSURE
- Site: Twenty days after intradermal induction, fur of the left flank (approximately 3 x 7 cm) of all animals was clipped with an electric clipper and further shaved with an electric shaver as closely as possible. On the next day, 3 sites (E to G) for challenge were provided for each animal and 0.1 mL of each substance for challenge test suspensions at 2 concentrations (50 and 25 % w/w) and polyethene glycol was applied via a patch 1.5 cm in diameter, which was fixed in place with a polyethylene film tape for 24 hours for occlusive application of challenge.
- Exposure period: 24 hours.
- Evaluation (hr after challenge): Observations of the application sites were made 24 hours and 48 hours after removal of the application for challenge and skin reactions were scored and recorded.

OBSERVATIONS
-Animals were observed for clinical signs once daily for the duration of the study (Day 0 to Day 24).
-Animals were weighed on the starting day (Day 0), the day of topical induction (Day 7), the day of challenge (Day 21) and the final observation day (Day 24).
-Observations of skin reactions were performed at 24 and 48 hours after removal of the challenge application. Skin reactions were scored according to the following:
0 = No visible change
1 = Discrete or patchy erythema
2 = Moderate and confluent erythema
3 = Intense erythema and swelling

EVALUATION
-For each application challenge site the severity of skin reaction was scored, the mean score for each test group at each observation time point was calculated and the positive reaction rate [(the number of animals with a score of 1 or above) / (the number of animals used) x 100] was determined. The sensitisation potential of the test material was assessed by comparison of the mean score and positive reaction rate between the test material and control groups.
-The test material was judged to be positive if the mean score and the positive reaction rate were higher in the test material group.
Challenge controls:
50 and 25 % for 24 hours exposure.
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole (MBT)
Positive control results:
No positive control group was provided in this study but 2-Mercaptobenzothiazole (MBT) study data has been conducted at the test facility. Clear sensitisation potential of MBT was observed and thus the experimental procedure used in the facility is appropriate.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50 % w/w
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 % w/w
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50 % w/w
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25 % w/w
No. with + reactions:
0
Total no. in group:
10

Preliminary studies

-Intradermal injection: Erythema of score 2 or 1 was observed at concentrations 25 and 10 % w/w and erythema score of 1 was observed at concentrations 5 and 1 % at 24 and 48 hours post administration in 2/2 or 1/2 animals, however necrosis was not observed. As a result the concentration for intradermal induction was set at 25 % w/w (maximum concentration with no necrosis).

-Topical induction / challenge: No skin reactions were observed at 50, 25, 10, 5, 2.5 or 1 % w/w at 24 or 48 hours after patch removal. As a result the concentration was set at the maximum applicable concentration of 50 % w/w for the topical induction.

Main Study Treatment Group

There were no skin reactions in the observation at 24 or 48 hours after removal of the application in any animal at 50 or 25 % test material dose. The mean score was therefore 0 and the positive reaction rate was 0 %. There was no reaction in any animal to the vehicle so positive reaction rate for the vehicle group was also 0 %.

Control group

There were no skin reactions in the observation at 24 or 48 hours after removal of the application in any animal so reaction rate was 0 % for both 25 and 50 % test material dosage.

Observations:

- Clinical observations: Animals were observed for clinical signs once daily from the starting day of induction (Day 0) to the end of skin observation after challenge (Day 24). During the study period, there were no abnormalities in clinical signs observed.

- Body weight: all animals were weighed on the starting day of induction (Day 0), day of topical induction (Day 7), day of challenge (Day 21) and the final day of observation (Day 24). At the final day of observation (Day 24), a body weight decrease in comparison with previous value (Day 21) was observed in 3/10 animals in the test material group.

Interpretation of results:
other: Not sensitising in accordance with EU criteria
Conclusions:
Under the conditions of the study, the test material is not considered to be a skin sensitiser.
Executive summary:

The skin sensitisation potential of the test material was determined in accordance with the standardised guideline OECD 406 using a guinea pig maximisation test.

The study was performed in female Hartley guinea pigs and a preliminary study was performed first to determine the test material concentrations suitable for use. For the intradermal injection the maximum concentration not causing necrosis was set at 25 % w/w and for the topical induction was set at 50 % which was the maximum physically applicable concentration. The challenge application was set at 50 % (the maximum non irritating concentration) and 25 % (half thereof). In the main study animals were subjected to intradermal application on Day 0 and on Day 6 they received a topical induction which was applied for 48 hours. The challenge was performed on Day 20 and was applied for 24 hours with observations at 24 and 48 hours post removal.

No skin reactions were observed in the test material treatment group after challenge at either 50 or 25 % w/w concentration, the mean score was 0 and the positive reaction rate was 0 %. The same results were seen in the control group. In clinical signs and bodyweight data there were no abnormalities that were thought to be attributable to application of the test material. The positive control data showed that the experimental procedure was valid.

Under the conditions of this study it was concluded that the test material had no skin sensitisation potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Guinea Pig Maximisation - Key Study (Hara, 2006)

The skin sensitisation potential of the test material was determined in accordance with the standardised guideline OECD 406 using a guinea pig maximisation test. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was performed in female Hartley guinea pigs and a preliminary study was performed first to determine the test material concentrations suitable for use. For the intradermal injection the maximum concentration not causing necrosis was set at 25 % w/w and for the topical induction was set at 50 % which was the maximum physically applicable concentration. The challenge application was set at 50 % (the maximum non irritating concentration) and 25 % (half thereof). In the main study animals were subjected to intradermal application on Day 0 and on Day 6 they received a topical induction which was applied for 48 hours. The challenge was performed on Day 20 and was applied for 24 hours with observations at 24 and 48 hours post removal.

No skin reactions were observed in the test material treatment group after challenge at either 50 or 25 % w/w concentration, the mean score was 0 and the positive reaction rate was 0 %. The same results were seen in the control group. In clinical signs and bodyweight data there were no abnormalities that were thought to be attributable to application of the test material. The positive control data showed that the experimental procedure was valid.

Under the conditions of this study it was concluded that the test material had no skin sensitisation potential.

Buehler Test - Supporting study (Ueno, 2000)

The potential of the test material to cause skin sensitisation in female Hartley guinea pigs was determined using the Buehler test. Three different lots of test material (1, 2 and 3) were tested. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

In each sensitising group of test material 1, test material 2, and test material 3, sensitisation was induced at 25 % concentration of each test material. Sensitisation was performed three times per week for nine doses. Challenge was treated with 25 and 10 % and petrolatum one day after the last sensitisation dose. For observation of skin sensitisation, any skin reaction was not seen as with controls which were induced sensitisation with petrolatum, and treated challenge with each test material sensitising group at the same concentrations.

In clinical signs and bodyweights, any anomaly due to the test substance was not observed in any test group during observation period.

Under the conditions of the study, three different lot numbers of the test material are not considered to be skin sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.