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Toxicological information

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:
From 2010-07-20 to 2010-09-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The Murine Local Lymph Node Assay (LLNA) is the first-choice method for in vivo testing according to the REACH Regulation. However, this reliable Buehler test was performed before entry into force of the REACH Regulation.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: no data
- Expiration date of the lot/batch: no data
- Purity test date: no data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: The test article was dosed as received for the induction and challenge.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was dosed as received for the induction and challenge.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elm Hill Breeding Laboratories
- Age at study initiation: 9 weeks (dose range); 4 weeks at start of dosing (Day 1) of the main study
- Weight at study initiation: 475-720 grams (dose range); 300-425 grams at outset (Day 1) of the main study
- Housing: Animals were individually housed upon receipt and upon assignment to study in compliance with USDA Guidelines. Calvert is a USDA registered and fully AAALAC accredited facility. The room in which the animals were kept was documented in the study records. No other species were kept in the same room.
- Diet (e.g. ad libitum): All animals had access to Harlan Teklad Guinea Pig Diet (certified) ad libitum
- Water (e.g. ad libitum): Tap water was available ad /ibitum, to each animal via an automatic watering device
- Acclimation period: Study animals were acclimated to their housing for a minimum of 5 and 7 days respectively for the Dose range and Main Assay prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19° to 25°C
- Humidity (%): 30 to 97%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% / 0.3 mL - applied to a Hilltop chamber (approximately 4-6 cm²)
Day(s)/duration:
Weeks 1-3: a total of three 6-hour occluded dermal applications of the test article
Adequacy of induction:
other: concentration chosen for induction was generally one that produces mild irritation
Challenge
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% / 0.3 mL - applied to a Hilltop chamber (approximately 4-6 cm²)
Day(s)/duration:
Fourteen days after the last induction
Adequacy of challenge:
other: highest concentration to be used was generally one in the vehicle used that induced no scores >= 1 and scores not exceeding two ± in the groupo of four animals
No. of animals per dose:
- 4 Primary Irritation (Dose Range)
- 20 Test Article Group (10 male/10 female)
- 10 Vehicle Control Group (5 male/5 female)
- 6 Positive Control Group (3 male/3 female)
Details on study design:
Range Finding Tests:
- Primary Irritation Screens: Prior to initiation of the main study, the irritation potential was determined. Each side of four naive animals were clipped free of hair the day prior to dose. Four concentrations of the test article (10%, 25%, 50% or 100%) were dosed on four separate sites. The location of each of the four applications of test article differed in each of the four animals to compensate for any site-to­site variations. For grading of the response, the procedure described below for primary challenge was used, except that only 24-hour grades were obtained. The concentration chosen for induction was generally one that produces mild irritation. The highest concentration to be used for challenge was generally one in the vehicle used that induces no scores > 1 and scores not exceeding two [±] in the group of four animals.

Main Assay:
- Site Preparation and Induction:
The sites of application on each animal were clipped free of hair with a small animal clipper the day prior to each induction and challenge application.
The test article or vehicle or positive control was applied to a 25 mm Hill Top Chamber® patch (Hill Top Research, Inc., Miamiville, Ohio). The animal was placed in a restrainer and the patch applied to the clipped surface as quickly as possible after the test article preparation has been applied to the patch. The patch was occluded with a rubber dental dam by pulling it tightly over the animal and fastening it to the bottom of the restrainer with binder clips. The restrainer was adjusted to minimize movement of the animal during the exposure period. Both edges of the dental dam will be under the front and back adjustable braces of the restrainer.
Six (± 30 min) hours later, the dental dam and patch were removed and the animal was placed in its cage. The test article preparations were removed by gently wiping with mineral oil and gauze before returning animals to their cages. The treated sites were examined after each dosing day and scored at 24 (± 4) and 48 (± 4) hours. This dosing and scoring procedure was done once a week for 3 weeks for a total of three 6-hour exposures to the test article.
- Challenge: Fourteen days after the last induction exposure, the animals were challenged in the same manner but the patches were applied to freshly clipped skin sites that have not been exposed previously. Since the vehicle used for induction was not water (mineral oil) and was the same for both induction and challenge, the test animals were also challenged with vehicle. The vehicle control group was also challenged with the test article and the vehicle. Twenty (± 2) hours after removal of the challenge patch, all animals were depilated with Nair Lotion Hair Remover. The depilatory was placed on the test sites and surrounding areas and left on for 8-12 minutes. The depilatory was then thoroughly washed off with water and animals patted dry and returned to their cages. The inside of each cage was wiped to remove any depilatory that might have contaminated the cage. A minimum of two hours after depilation, test sites were graded such that the grading was done 24 (± 1) hours after removal of the challenge patch (24- hour grade). The grading was repeated 24 (± 2) hours later (48-hour grade).
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene (DNCB)

Results and discussion

Positive control results:
The positive control group, induced and challenged with DNCB, exhibited the anticipated positive responses at challenge, indicating that the methods employed in this study were valid.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100% Butyl Benzoate
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100% Butyl benzoate
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Mineral oil
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
One control animal was found dead prior to scording (Day 29) - necropsy revealed the intestines to be dark red and empty; no signs of systemic toxicity and all surviving animals gained weight during the study
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Mineral oil
No. with + reactions:
0
Total no. in group:
9
Clinical observations:
One control animal was found dead prior to scording (Day 29) - necropsy revealed the intestines to be dark red and empty; no signs of systemic toxicity and all surviving animals gained weight during the study
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100% Butyl Benzoate
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100% Butyl Benzoate
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Mineral oil
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Mineral oil
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.2% DNCB
No. with + reactions:
6
Total no. in group:
6
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.2% DNCB
No. with + reactions:
6
Total no. in group:
6
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, induction with the test substance at 100% did not elicit a delayed contact hypersensitivity response in guinea pigs when challenged at 100%.