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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:
1st October to 4th December 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted to GLP. Background irritation was observed in the naive control animals.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

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:
Study older than 2002 when TG LLNA adopted

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: slightly viscous, clear, brown liquid
- Expiration date of the lot/batch: 01-09-1997
- Storage condition of test material: 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: U.S.D.A. licensed supplier
- Age at study initiation: All test and primary challenge naive control animals were 6-11 weeks old (±5 days).
- Weight at study initiation: At the start of the induction phase of testing the test and primary challenge naive control animals weighed 370-481 grams. At the start of the re-challenge phase, the re-challenge naive control animals weighed 438-703 grams.
- Housing: Animals were individually housed in wire mesh suspension cages.
- Diet/water (e.g. ad libitum): Teklad Guinea Pig Diet and tap water were supplied ad libitum during both acclimation and test periods.
- Acclimation period: Prior to use, all animals were acclimated for at least seven days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79° F
- Humidity (%): 30-70%.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
Pilot phase: undiluted test material and 50%, 25%, 10%, 5%, 2.5%, 1%, and 0.5% of the test material in mineral oil.
Induction phase: 2.5% of the test material in mineral oil.
Challenge phase: 1% of the test material in mineral oil.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
Pilot phase: undiluted test material and 50%, 25%, 10%, 5%, 2.5%, 1%, and 0.5% of the test material in mineral oil.
Induction phase: 2.5% of the test material in mineral oil.
Challenge phase: 1% of the test material in mineral oil.
No. of animals per dose:
A total of 48 animals were utilized. Twenty test animals, twenty naive control animals, and eight pilot animals were used. Equal numbers of males and females were included in each animal group. The ten primary challenge naive control animals were common to this study.
Details on study design:
RANGE FINDING TESTS:
The irritation phase had the purpose of determining the proper level of test material to be used in the induction and challenge phases. The irritation potential of the test material at levels of undiluted, 50%, 25%, 10%, 5%, 2.5%, 1%, and 0.5% was evaluated in two groups of four animals each. Four levels of the test material were evaluated per animal such that each animal in a given pilot group was exposed to the same levels. Dilutions of the test material were formulated w/v in Mineral Oil (supplied by Chevron). The position of the different concentrations of the test material on the animals was varied to adjust for possible site-to-site variation in response.

The day prior to test material exposure, the hair was removed from each of the animal's backs using a small animal clipper. Closed patches were applied to the animals in the following manner: A 0.3 ml quantity of each test preparation was applied into a 25 mm Hill Top Chamber®. The animal was placed into the restrainer, and the chambers were applied to the clipped surface as quickly as possible. The chambers were occluded with rubber dental dam pulled taut and fastened to the bottom of the restrainer with clips. The restrainer was adjusted to minimize movement of the animal during exposure. Approximately six hours later, the dental dam and chambers were removed, and the animal was taken from the restrainer and placed in its cage. The day following the irritation exposure all animals were depilated and scored.

MAIN STUDY
A. INDUCTION EXPOSURE
The purpose of this phase was to dermally expose the animals so that, if the material was a sensitizer, the physiological process required to ultimately allow the generation of an immunological response could be initiated.

The left shoulder of each animal was clipped with a small animal clipper the day before exposure.
The animals were restrained, and a 0.3 ml quantity of the test preparation was applied using a Hill Top Chamber® as previously described above. The procedure was repeated at the same site once a week for the next two weeks for a total of three approximate six-hour exposures (the interval between induction exposures was seven days). After the last induction exposure, the animals were left untreated for approximately two weeks (14 days) before primary challenge.

B. CHALLENGE EXPOSURE
The purpose of this phase was to investigate the elicitation of response. The test animals, which had three previous exposures to the test material at appropriate intervals, were again exposed in the challenge phase approximately two weeks after the last induction exposure. In addition, ten naive animals, which had never been exposed to the test material, are concurrently treated with the same test material concentration.

The same exposure procedure as for the "Induction Phase" was used except the chambers were applied to a skin site that had not been exposed previously.

OTHER:
Re-challenge Phase:
The purpose of this phase was to confirm the responses noted during the primary challenge phase. The test animals, which had previous exposures to the test material at appropriate intervals, were again exposed in the re-challenge phase approximately one week after the primary challenge exposure. In addition, ten naive animals, which had never been exposed to the test material, were concurrently treated with the same test material concentration.

The same exposure procedure as for the "Primary Challenge Phase" was used, except the chambers were applied to a skin site that had not been exposed previously.
Challenge controls:
yes
Positive control substance(s):
yes

Results and discussion

Positive control results:
Group 1 test animals received one induction treatment utilizing 1-CHLORO-2,4-DINITROBENZENE (DNCB) at a concentration of 0.3% formulated w/v in 95% ethanol. Approximately two weeks (14 days) following the induction treatment, a primary challenge treatment was conducted utilizing the ten test animals and five naive control animals. Each animal received 1-CHLORO-2,4-DINITROBENZENE (DNCB) at concentrations of 0.1%, 0.05%, and 0.01% formulated w/v in acetone.

Group 2 test animals received three induction treatments at weekly intervals utilizing a - Hexylcinnamaldehyde, tech., 85% at a concentration of 2.5% formulated w/v in 95% ethanol. Approximately two weeks (16 days) following the induction treatments, a primary challenge treatment was conducted utilizing the ten test animals and five naive control animals. Each animal received a - Hexylcinnamaldehyde, tech., 85% at concentrations of 5%, 2.5%, and 1.0% formulated w/v in acetone.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1% w/v in mineral oil
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
1 animal with slight but confluent, or moderate patchy erythema (1) and 9 animals with slight, patchy erythema (±)
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1% w/v in mineral oil. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: 1 animal with slight but confluent, or moderate patchy erythema (1) and 9 animals with slight, patchy erythema (±).
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1% w/v in mineral oil
No. with + reactions:
1
Total no. in group:
19
Clinical observations:
1 animal with slight but confluent, or moderate patchy erythema (1) and 18 animals with slight, patchy erythema (±) and 1 animal was found dead.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1% w/v in mineral oil. No with. + reactions: 1.0. Total no. in groups: 19.0. Clinical observations: 1 animal with slight but confluent, or moderate patchy erythema (1) and 18 animals with slight, patchy erythema (±) and 1 animal was found dead..
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
1% w/v in mineral oil
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
3 animals with slight but confluent, or moderate patchy erythema (1) and 17 animals with slight, patchy erythema (±)
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 1% w/v in mineral oil. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: 3 animals with slight but confluent, or moderate patchy erythema (1) and 17 animals with slight, patchy erythema (±).
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1% w/v in mineral oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
10 animals with slight, patchy erythema (±)
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1% w/v in mineral oil. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: 10 animals with slight, patchy erythema (±).
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
1% w/v in mineral oil
No. with + reactions:
1
Total no. in group:
19
Clinical observations:
1 animal with slight but confluent, or moderate patchy erythema (1) and 18 animals with slight, patchy erythema (±) and 1 animal was found dead.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1% w/v in mineral oil. No with. + reactions: 1.0. Total no. in groups: 19.0. Clinical observations: 1 animal with slight but confluent, or moderate patchy erythema (1) and 18 animals with slight, patchy erythema (±) and 1 animal was found dead..
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
1% w/v in mineral oil
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
3 animals with slight but confluent, or moderate patchy erythema (1) and 17 animals with slight, patchy erythema (±)
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 1% w/v in mineral oil. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: 3 animals with slight but confluent, or moderate patchy erythema (1) and 17 animals with slight, patchy erythema (±).

Any other information on results incl. tables

See attached illustration.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test material was not considered a sensitizer in the Hartley guinea pig.
Executive summary:

The potential of the test material as a 2.5% w/v in Mineral Oil to produce delayed contact hypersensitivity in guinea pigs was evaluated using an adaptation of the method of Ritz and Buehler. Following primary challenge using the test material, as a 1% w/v formulation in Mineral Oil, the incidence of grade 1 responses in the test group (2 of 19) was compared to that of the naive control group (1 of 10). The incidence and severity of these responses in the test group were essentially comparable to those produced by the naive control group suggesting that sensitization had not been induced. However one test animal responded with a grade ± at the 24 hour reading which increased to a grade 1 at the 48 hour reading. This type of response is suspect as a sensitization type response. Therefore, a re-challenge was conducted to clarify the response noted during primary challenge.

Following re-challenge using the test material, as a 1% w/v formulation in Mineral Oil, the incidence of grade 1 responses in the test group (5 of 19) was compared to that of the naive control group (2 of 10). The incidence and severity of these responses in the test group were again essentially comparable to those produced by the naive control group. The failure of the test animals to exhibit a higher incidence of responses over that of the naive control group indicates that the responses noted are due to irritation and not sensitization. Therefore, it can be concluded that sensitization had not been induced.