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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:
December 20, 1994 - January 27, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
Version / remarks:
Pesticide Assessment Guidelines, Subdivision F: Hazard Evaluation: Human and Domestic Animals, Section 81-6 OECD Guideline 406, Buehler Test
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The Buehler Test (1995) met the previous requirements before the entry into force of REACH. The Buehler Test is suitable and reliable to cover this endpoint. For this reason and for animal welfare reasons, no further in vivo study (LLNA test) needs to be performed.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Davidson's Mill Farms, South Brunswick, NJ
- Weight at study initiation: 335 – 388 g (males), 288 - 413 g (females)
- Housing: The animals were group housed in suspended stainless steel caging with mesh floors.
- Diet: Pelleted Purina Guinea Pig Chow
- Water: ad libitum (tap water)
- Acclimation period: 6 or 7 days
- Indication of any skin lesions: Only healthy animals without pre-existing skin irritation were selected for test.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 22.2
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
50% and 75% (0.4 mL)
Day(s)/duration:
Two times each week (6 hours) for three weeks
Adequacy of induction:
other: Due to the severity of irritation noted at all test sites, the concentration was reduced to 50% after the second induction phase .
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
20%
Day(s)/duration:
Thirteen days after last induction/ 6 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
20%
Day(s)/duration:
Seven days after challenge #1
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Number of animals in test group: 20
Number of animals in negative control group: 10
Details on study design:
RANGE FINDING TESTS: The fur was removed by clipping the dorsal area and flanks of each guinea pig. This area was divided into four test sites (two sites on each side of the midline) on each animal. The test substance was applied neat (100%) and was also diluted with distilled water to yield concentrations of 75%, 50%, 25%, 20%,10%, 5% and 2%. Each concentration was applied to a test site using an occlusive 25 mm Hilltop Chamber. The sites were wrapped with non-allergenic Durapore adhesive tape. After 6 hours of exposure, the chambers were removed. Twenty-four hours after application, each site was evaluated for local reactions (erythema). Based on these results, the highest non-irritating concentration selected for the challenge phase was a 20% w/w suspension in distilled water. Furthermore, the selected highest non-irritating concentration of the positive control (1-Chloro-2,4-Dinitrobenzene (DNCB)) was determined to be a 0.04% w/w solution in acetone.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 6
- Exposure period: 6 hours
- Test groups: treatment with the test item
- Site: left side of each animal
- Frequency of applications: two times per week
- Duration: three weeks
- Concentrations: 75% test item in water (concentration was reduced to 50% after the second induction phase)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: thirteen days after last induction
- Exposure period: 6 hours
- Test groups: treatment with test item
- Control group: ten animals were treated with 20% test item suspension
- Site: right site of each animal
- Concentrations: 20% test item in water
- Evaluation (hr after challenge): 24 and 48

C. RECHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: seven days after challenge
- Exposure period: 6 hours
- Test groups: treatment with test item
- Control group: An additional group of ten animals was placed on test to serve as a naive group for rechallenge and treated with the test item suspension.
- Site: naive right site of each animal
- Concentrations: 20% test item in water
- Evaluation (hr after challenge): 24 and 48
Challenge controls:
An additional group of ten animals was placed on test to serve as a naive group for rechallenge.
Positive control substance(s):
yes
Remarks:
1-Chloro-2,4-Dinitrobenzene

Results and discussion

Positive control results:
INDUCTION PHASE
Positive Control Animals (0.08% DNCB in 80% aqueous ethanol): Very faint to severe erythema (0.5-3) was exhibited at all positive control sites during the induction phase. Eschar and/or desquamation were evident at several sites after the third and fifth inductions. Due to the severity of irritation, the dose sites were relocated to an adjacent area for inductions four and six.

CHALLENGE PHASE
Positive Control Animals (0.04% DNCB in acetone): Seven of twenty positive control animals exhibited signs of a sensitization response (faint to moderate erythema (1-2)) 24 hours after challenge. Similar indications persisted in three of these animals through 48 hours. Very faint erythema (0.51) was noted at most other positive control sites following the challenge dose.

Positive Naive Control Animals (0.04% DNCB in acetone); Very faint erythema (0.5) was noted at Three of ten positive naive control animals 24 hours after challenge. Irritation cleared from all affected sites by 48 hours.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
20 %
No. with + reactions:
1
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
20 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
20 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
20 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
20 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
20 %
No. with + reactions:
0
Total no. in group:
10

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the findings of the Dermal Sensitization test (Buehler method) the test item is not considered to be a skin sensitizer.
Executive summary:

A Dermal Sensitization test was conducted with guinea pigs to determine the potential for the test substance to produce sensitization after repeated topical applications.

The test substance was applied to twenty healthy test guinea pigs and Dinitrochlorobenzene (DNCB, 0.08%), respectively, was applied to twenty healthy positive control animals two times each week for a three week induction period. Thirteen days after the last induction, a challenge dose of the test and positive control substances at their highest non-irritating concentrations (HNIC, 20% and 0.04%, respectively) was applied to a naive site on each guinea pig. Approximately

24 and 48 hours after each induction and challenge dose, the animals were scored for erythema.

Two naive control groups (ten animals for the test substance and ten for the positive control substance) were maintained under the same environmental conditions and treated with the test or positive control substance at challenge only.

Very faint to severe erythema (score 0.5-3), was noted at all test sites during the induction phase. Irritation was most severe following the second, third and fifth inductions. Eschar and/or desquamation were also evident at several sites following these doses. A reduction in the severity of irritation was noted shortly after the induction concentration was reduced (75% to 50% suspension) or after the dose sites were relocated to an adjacent area.

Very faint 10 severe erytheme (score 0.5-3) was exhibited at all positive control sites during the induction phase. Eschar and/or desquamation were evident at some sites after the third and fifth inductions. Due to the severity of irritation, the dose sites were relocated to an adjacent area for inductions four and six.

Faint erythema (score 1) was noted at one test site 24 hours after the challenge dose. Very faint erythema (score 0.5) was noted at most other sites 24 and 48 hours after challenge.

Very faint erythema (score 0.5) was noted at several naive control lest sites alter the challenge

dose.

Seven of twenty positive control animals exhibited signs of a sensitization response (faint to moderate erythema (score 1-2)) 24 hours after challenge. Similar indications persisted in three of these animals through 48 hours. Very faint erythema (score 0.5) was noted at most other positive control sites following the challenge dose. Very faint erythema (score 0.5) was noted at three of ten positive naive control animals 24 hours after challenge. Irritation cleared from all affected sites by 48 hours,

Due to the presence of taint erythema (score 1) at one test site 24 hours after challenge, a rechallenge was conducted. An additional group of ten animals was placed on test to serve as a naive control group for rechallenge.

Very faint (score 0.5) erythema was noted at five of twenty test sites after rechallenge. Erythema cleared from two of the five affected sites by 48 hours.

 

Very faint erythema (score 0.5) was noted at three naive control sites 24 and 48 hours after rechallenge.

Based on the results of testing, the test substance is not considered to be a contact sensitizer.