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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
Nine induction exposures, and reduced number of animals (10 and 5 instead of 20 and 10 for treated and control, respectively)
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
Study was performed before LLNA was formally validated and available as OECD guideline in 2002.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Triclosan
- Physical state: white crystalline powder
- Analytical purity: 99.7%
- Lot/batch No.: #5.2.0211.0
- Stability under test conditions: stable at room temperature for 5 years
- Storage condition of test material: at room temperature

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
- Age at study initiation: young adult animals were used
- Weight at study initiation: males 305-345 g, females 304-361 g
- Housing: group housed in suspended stainless steel caging with mesh floors
- Diet (e.g. ad libitum): Pelleted purina Guinea Pig Chow, ad libitum
- Water (e.g. ad libitum): Filtered tap water, ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22 °C
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
For induction, a test dose of 25% test item in propylene glycol was initially selected, however, due to the severity of irritation after the second and fourth application, the induction concentration was reduced to 10% for inductions 3 and 4, and further decreased to 2% for inductions 5 through 9.
For challenge, a dose of 5% test item in propylene glycol was selected on the basis on a preliminary range-finding test.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
propylene glycol
Concentration / amount:
For induction, a test dose of 25% test item in propylene glycol was initially selected, however, due to the severity of irritation after the second and fourth application, the induction concentration was reduced to 10% for inductions 3 and 4, and further decreased to 2% for inductions 5 through 9.
For challenge, a dose of 5% test item in propylene glycol was selected on the basis on a preliminary range-finding test.
No. of animals per dose:
10
Details on study design:
RANGE FINDING TESTS:
Prior to testing for sensitisation, a pre-test was conducted for determination of the primary irritation of the test material. For this purpose, the fur was removed by clipping the dorsal area and flanks of each test animal. This area was divided into 4 test sites and the test substance was diluted with propylene glycol to yield concentrations of 95%, 75%, 50%, 25%, 10%, 7%, 5%, 2% and 1%. 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 skin reactions (erythema). From these results, the highest concentration that produced responses in 4 guinea pigs no more severe than 2 scores of 0.5 and 2 scores of zero was established and used for challenge. Based on these findings, the selected concentration for challenge was 5% solution ot test item in propylene glycol.

MAIN TEST:
On the day before initiation, the fur of the test animals was removed by clipping the dorsal area and flanks.
A-INDUCTION
Initially, a test dose of 25% test item in propylene glycol was selected for induction; however, due to the severity of irritation after the second and fourth application, the induction concentration was reduced to 10% for inductions 3 and 4, and further decreased to 2% for inductions 5 through 9.
The test substance was applied onto the skin of each test animal 3 times each week for a 3 week induction period total of 9 applications). The application site was maintained under occlusive conditions for an exposure period of 6 hours; after removal of the dressing, the application sites were gently wiped with 95% ethanol and a clean tewel to remove any residual substance.
B-CHALLENGE
Twelve days after the last induction, a challenge dose of the test item (5% in propylene glycol) was applied to a naive site on each guinea pig, similarly as described above. These sites were evaluated for a sensitization response (erythema) at 24 and 48 hours after the challenge application.

READING:
Approximately 24 and 48 hours after each induction and challenge dose, the application sites were examined for skin reactions, and the findings were scored according to following system:
0 = no reaction
0.5 = very faint erythema, usually non-confluent
1 = faint erythema usually confluent
2 = moderate erythema
3 = severe erythema with or without edema

EVALUATION OF THE SENSITIZING RESPONSE:
In order to evaluate the sensitization response, 2 indices were used; one for incidence and one for severity for both test and positive control animals.

The incidence index was calculated to evaluate the incidence of erythema (sensitization response) 24 and 48 hours after challenge according to the following formula: lncidence Index = Number of erythema scores> 0.5/Number of animals evaluated

The severity index (sensitization produced) at 24 and 48 hours after challenge was calculated using the following formula:
Severity Index = Sum of erythema scores /Number of animals evaluated

The following criteria were used to classify the test substance as a potential contact sensitizer:
1 - At the 24 and/or48 hour scoring interval, 20% or more (i.e. incidence index of 0.2 or more) of the test animals exhibit a positive response (scores>
0.5) in the absence of similar results in the naive control group.
2 - The positive reaction must persist to 48 hours in at least one test animal
Challenge controls:
Two naïve control groups (5 animals for the test substance and 5 for the positive control) were maintained under the same environmental conditions and treated with the test or positive control substance at challenge only.
Positive control substance(s):
yes
Remarks:
1-Chloro-2,4-Dinitrobenzene (DNCB)

Results and discussion

Positive control results:
Positive Control Animals (0.03% DNCB in acetone): Twenty-four hours after challenge, all control sites exhibited signs of a sensitization response (faint to severe erythema; these reactions persisted to 48 hours in all animals.
Positive Naive Control Animals (0.03% DNCB in acetone): Very faint erythema was noted at 3 of 5 positive naive control sites 24 hours after challenge. Irritation persisted to 48 hours in 2 guinea pigs.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5% test item in propylene glycol
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
very faint erythema graded 0.5 was reported for the 6 test sites
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5% test item in propylene glycol. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: very faint erythema graded 0.5 was reported for the 6 test sites.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5% test item in propylene glycol
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
the findings as decribed above persisted in 5 cases
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5% test item in propylene glycol. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: the findings as decribed above persisted in 5 cases.
Reading:
1st reading
Hours after challenge:
48
Group:
other: naive test animals treated with the test item at challenge only.
Dose level:
5% test item in propylene glycol
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
very faint erythema graded 0.5 was noted in 2 animals
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: other: naive test animals treated with the test item at challenge only.. Dose level: 5% test item in propylene glycol. No with. + reactions: 2.0. Total no. in groups: 5.0. Clinical observations: very faint erythema graded 0.5 was noted in 2 animals.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.03% DNCB in acetone
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
all animals showed signs indicative of skin sensitisation (faint to severe erythema)
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.03% DNCB in acetone. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: all animals showed signs indicative of skin sensitisation (faint to severe erythema).
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.03% DNCB in acetone
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
reaction similar as seen after 24 hours
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.03% DNCB in acetone. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: reaction similar as seen after 24 hours.
Reading:
1st reading
Hours after challenge:
24
Group:
other: positive naive control treated with DNCB at challenge only
Dose level:
0.03% DNCB in acetone
No. with + reactions:
3
Total no. in group:
5
Clinical observations:
very faint erythema graded 0.5 was seen in 3 cases
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: other: positive naive control treated with DNCB at challenge only. Dose level: 0.03% DNCB in acetone. No with. + reactions: 3.0. Total no. in groups: 5.0. Clinical observations: very faint erythema graded 0.5 was seen in 3 cases.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: positive naive control treated with DNCB at challenge only
Dose level:
0.03% DNCB in acetone
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
the findings seen after 24 hours persisted in 2 cases to 48 hours
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: positive naive control treated with DNCB at challenge only. Dose level: 0.03% DNCB in acetone. No with. + reactions: 2.0. Total no. in groups: 5.0. Clinical observations: the findings seen after 24 hours persisted in 2 cases to 48 hours.

Any other information on results incl. tables

INDUCTION:

All test sites exhibited very faint to severe erythema during the induction phase.

Edema, desquamation, hyperkeratosis and/or eschar were noted at many sites.

Due to the severity of irritation after the second and fourth inductions, the induction concentration of the test substance was reduced from a 25% solution in propylene glycol to a 10 and then a 2% solution. A decrease in the overall severity of irritation was noted following each reduction in induction concentration.

CHALLENGE:

Twenty-four hours after challenge, very faint erythema was noted at 6 test sites. Irritation persisted in 5 animals through 48 hours. Although no irritation was noted at any naive sites 24 hours after challenge, 2 naive animals exhibited very faint erythema at 48 hours.
All positive control sites exhibited very faint to severe erythema during the induction phase. Eschar, hyperkeratosis and/or desquamation were evident at all sites. Twenty-four and 48 hours after challenge, all positive control sites exhibited signs of a sensitization response (faint to severe erythema). Twenty hours after challenge, very faint erythema was noted at 3 of 5 positive naive control sites.
Irritation persisted to 48 hours in 2 affected animals.

CLINICAL SYMPTOMS:

All animals gained weight and no signs of gross toxicity, adverse pharmacologic effects or abnormal behaviour were reported.

Applicant's summary and conclusion