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Diss Factsheets

Administrative data

Description of key information

 Two reliable Delayed Contact Hypersensitivity in Guinea Pigs studies (Buehler Method) according to OECD Guideline 406 are available. The test item was observed to be sensitising to the skin in both.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006-08-02 to 2006-09-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
not specified
Remarks:
The study is performed before 2008, with report available in 2012.
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.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: E-015/2005-1
- Purity test date: 2005-10-24
- Purity: 99.01 wt% (GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: no data



Species:
guinea pig
Strain:
Hartley
Remarks:
Hsd: POC DH
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan
- Age at study initiation: 4 weeks at start of dosing
- Weight at study initiation: 300-366 grams
- 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: minimum of 7 days prior to dosing
- Indication of any skin lesions: not indicated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19° to 26°C
- Humidity (%): 45 to 80%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
concentration:
100%
volume:
0.3 mL per site
Day(s)/duration:
weeks 1 - 3: a total of three 6-hour exposures to the test item
Adequacy of induction:
other: concentration chosen for induction was generally one that produces mild irritation
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
concentration:
80%
volume: 0.3 mL per site
Day(s)/duration:
weeks 1 - 3: a total of three 6-hour exposures to the test item
Adequacy of induction:
other: concentration chosen for induction was generally one that produces mild irritation
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
concentration:
70%
volume:
0.3 mL per site
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:
primary irritation test: 4; secondary screen: 4; test article: 20 (10m, 10f); positive control: 6 (3m, 3f); vehicle control: 10 (5m, 5f)
Details on study design:
RANGE FINDING TESTS:
Dose-Range-Finding Study
a) Primary Irritation Screens: Prior to initiation of the main study, the irritation potential was determined. Four naive animals were exposed to four different concentrations of the test material by the patching technique described in site preparation and treatment. The location of each of the four concentrations of test article differed in each of the four animals to compensate for any site-to-site 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 > = 1and scores not exceeding two ± in the group of four animals.
b) Secondary Irritation Screen: An additional irritation screen was performed using four additional animals. Doses of 60%, 70%, 80% and 90% were investigated to choose the second and third induction doses and the challenge dose.
The dose chosen for induction: 0.3 ml
The dose chosen for challenge: 0.3 ml

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Test groups: 80% and 100% of test material in physiological saline
- Control group: vehicle only
- Frequency of applications: once a week
- Duration: 6 h
- Concentrations: 80% and 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Exposure period: 6 hours
- Test groups: 70% of test material
- Control group: vehicle only
- Concentrations: 70%

Positive control substance(s):
yes
Remarks:
1-chloro-2, 4-dinitrobenzene (DNCB) (induction: 0.3% in 80% ethanol; challenge: 0.2% in acetone)
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.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
70%
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
70%
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.2%
No. with + reactions:
6
Total no. in group:
6
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.2%
No. with + reactions:
6
Total no. in group:
6
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
70% test item
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
70% test item
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of this study, induction with the test substance at 100 and 80% did elicit a delayed contact hypersensitivity response in guinea pigs when challenged with the test article at 70%.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2007-09-27 to 2007-11-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
E-015/2005-1
- Purity test date:
2005-10-24
- Purity: 99.01 wt% (GC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature
- Solubility and stability of the test substance in the solvent/vehicle:
no data




Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hartley
- Age at study initiation: 5 weeks
- Weight at study initiation: 322-428 grams at the outset (Day 1) of the study; naïve controls: 462 - 726 grams (Day 35)
- 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 libitum, to each animal via an automatic watering device
- Acclimation period: Minimum of 7 days prior to dosing.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18° to 24°C
- Humidity (%): 24 to 75%
- Air changes (per hr): not indicated
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
concentration: 60%
volume: 0.3 mL per site
Day(s)/duration:
weeks 1 - 3 (once per week)
Adequacy of induction:
other: concentration for induction was generally one that produced mild irritation
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
concentration: 50%
volume: 0.3 mL per site
Day(s)/duration:
weeks 1 - 3 (once per week)
Adequacy of induction:
other: concentration for induction was generally one that produced mild irritation
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
concentration: 40%
volume: 0.3 mL per site
Day(s)/duration:
fifth week (once)
Adequacy of challenge:
other: highest concentration for challenge was generally one in the vehicle used that induced no scores >=1 and scores not exceeding two ± in the group of four animals
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
concentration: 30%
volume: 0.3 mL per site
Day(s)/duration:
sixt week (once) - rechallenge 6 days after primary challenge
Adequacy of challenge:
not specified
No. of animals per dose:
primary irritation: 4 (dose range); test article group: 20; vehicle control group: 10; positive control group: 6 and naïve control group: 4
Details on study design:
RANGE FINDING TESTS:
Primary Irritation Screens: Prior to initiation of the main study, the irritation potential was determined. Four naive animals were exposed to four different concentrations of the test material by the patching technique described in site preparation and treatment. The location of each of the four concentrations of test article differed in each of the four animals to compensate for any site-to-site 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 STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 per animal
- Exposure period: 6-hour exposures
- Test groups: 60% and 50% of test material
- Control group: vehicle

B. CHALLENGE EXPOSURE
- No. of exposures: 3 per animal
- Exposure period: 6-hour exposures
- Test groups: 60% and 50% of test material
- Control group: saline and 40% test article

OTHER:
In the sixth week a rechallenge was performed with a dose of 30%.
Positive control substance(s):
yes
Remarks:
1-chloro-2, 4-dinitrobenzene (DNCB) (induction: 0.3% in 80% ethanol; challenge: 0.2% in acetone)
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.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
40%
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
40%
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
40%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
40%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
saline
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
saline
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
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
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
Reading:
rechallenge
Hours after challenge:
24
Group:
other: naïve
Dose level:
none
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Reading:
rechallenge
Hours after challenge:
48
Group:
other: naïve
Dose level:
none
No. with + reactions:
0
Total no. in group:
4
Clinical observations:
no signs of systemic toxicity and all animals gained weight during the study
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of this study, induction with the test substance at 60% and 50% elicited a delayed contact hypersensitivity response in guinea pigs when challenged at 40%. The animals responded with the minimum number of responses required at challenge to meet the criteria of a sensitizer. Rechallenge of the test substance at 30% did not elicit a delayed contact hypersensitivity response in guinea pigs. The animals did not respond with the minimum number of responses required at rechallenge to meet the criteria of a sensitizer. Overall, it can be concluded that the test substance is a sensitizer.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The purpose of the two Delayed Contact Hypersensitivity in Guinea Pigs (Buehler Method) studies (according to OECD guideline 406) was to determine if the test article elicits a delayed dermal contact hypersensitivity response in guinea pigs by the measurement of skin reactivity.

 

In the first study, a dose range was initially performed in four naive animals at 10%, 25%, 50% and 100%. Based on these results and the scores seen at first induction (100%), a secondary screen was performed in four additional naïve animals at 60%, 70%, 80% and 90%. Based on these results, the first induction dose of 100% was reduced to 80% for the second and third induction. For challenge a dose of 70% was utilized for this study.

For the induction phase of the study, twenty guinea pigs (10/sex) in the test article group were induced with three six-hour occluded dermal applications of test substance (1st induction at 100% and 2nd and 3rd induction at 80% in saline). A vehicle group of ten animals (5/sex) was induced in the same manner with saline. A positive control group of six animals (3/sex) was induced with a known dermal sensitizer: 1-chloro-2, 4-dinitrobenzene (DNCB).

Fourteen days after the last induction, all animals were dermally challenged with occluded applications at naive test sites.

 Animals in the test article and vehicle control group were challenged with test substance (70%) in saline.

Animals in the positive control group were challenged with DNCB (0.2% in acetone). On the day following the challenge, all animals were depilated and approximately four hours later were scored for dermal irritation (24 hour).

Scoring was repeated at 48 hours. Under the conditions of the study, induction with test substance at 100% and 80% did elicit a delayed contact hypersensitivity response in guinea pigs when challenged with the test article at 70%.

In the second study, a dose range was performed in four naïve animals at 40%, 50%, 60% and 70%. Based on these results, an induction dose of 60% was chosen for this study. Based on observations made following the second induction the concentration for the third induction was lowered to 50% and moved to the right shoulder. The challenge dose chosen was 40%.

For the induction phase of this study, twenty guinea pigs (10/sex) in the test article group were induced with three six-hour occluded dermal applications of test substance (60% for 1st and 2nd induction and 50% for the 3rd induction). A vehicle group of ten animals (5/sex) was induced in the same manner with saline. A positive control group of six animals (3/sex) was induced with a known dermal sensitizer: 1-chloro-2,4-dinitrobenzene (DNCB). Fourteen days after the last induction, all animals were dermally challenged with occluded applications at naive test sites. Animals in the test article and vehicle control group were challenged with test substance (40%). Animals in the positive control group were challenged with DNCB (0.2% in acetone). On the day following the challenge, all animals were depilated and approximately four hours later were scored for dermal irritation (24 hour). Scoring was repeated at 48 hours.

Due to the results observed in the treated group, the Sponsor was contacted and a rechallenge was requested to be performed. Four naïve animals were added to the study at this point for the rechallenge.

Under the conditions of the study, induction with the test substance at 60% and 50% elicited a delayed contact hypersensitivity response in guinea pigs when challenged at 40%. The animals responded with the minimum number of responses required at challenge to meet the criteria of a sensitizer. Rechallenge of test substance at 30% did not elicit a delayed contact hypersensitivity response in guinea pigs. The animals did not respond with the minimum number of responses required at rechallenge to meet the criteria of a sensitizer. Overall, it was concluded that the test substance is a sensitizer.

 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results, the test substance is classified as sensitizing to the skin (category 1B) according to CLP regulation.