Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 26, 2001 to January 18, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted following internationally accepted testing guidelines and performed according to the GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
Buehler test

Test material

Constituent 1
Reference substance name:
Lysozyme, hydrochloride
EC Number:
232-954-0
EC Name:
Lysozyme, hydrochloride
Cas Number:
9066-59-5
Molecular formula:
C616H963N193O182S10 x HCl
IUPAC Name:
Lysozyme, hydrochloride

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Justification of Species: the guinea pig is conventionally used in skin sensitization studies to provide information on which human hazard can be judged, and is preferred by the regulatory agencies.
- Source: Charles River Laboratories; Wilmington, MA.
- Weight at study initiation: males 380-542 g; females 413-520 g
- Housing: suspended, wire bottom, stainless steel. 1-4 per cage (males separate from females).
- Diet: PMI Feeds, Inc.TM Guinea Pig Diet #5025; available ad libitum.
- Water: municipal water supply analyzed by TNRCC Water Utilities Division; available ad libitum from water bowls.
- A uarantine: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature:20 ± 3 °C
- Humidity: 30-70 %
- Air changes: 10-12 air changes/hour
- Photoperiod: 12-hour light/dark cycle

IN-LIFE DATES
Animlas were received by the laboratory at November 26 and December 10, 2001. They were treated with the test substance for first induction on December 19, for second induction on January 02 and for challenge on January 16, 2002. The in-life portion of the study was termined on January 02, 2002.

No contaminants were expected to have been present in the feed or water which would have interfered with or affected the results of the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Induction: 400 mg moistened w/0.5 ml corn oil
Challenge: 400 mg moistened w/0.5 ml corn oil
Challenge
Concentration / amount:
Induction: 400 mg moistened w/0.5 ml corn oil
Challenge: 400 mg moistened w/0.5 ml corn oil
No. of animals per dose:
2 males and 2 females (Range-finding)
15 males and 15 females (definitive, of which 10 treated and 5 used as naive control)
Details on study design:
IRRITATION SCREENING
Two male and two female albino guinea pigs were selected for irritation screening to determine both the maximum dose producing no more than moderate irritation, and the maximum non¬irritating dose. Tested in the screening were 400 mg of test substance moistened wich com oil, and 75 %, 50 % and 25 % w/v concentrations of the test substance in corn oil.

PREPARATION of ANIMALS
On the day prior to each treatment, the animals were prepared by clipping the back of the trunk free of hair to expose a longitudinal area at least 8 x 10 cm of each animal. Individual body weights were recorded on Days 0 and 28.

MAIN STUDY
A. INDUCTION EXPOSURE
- Application: for each induction treatment, animals were treated by introducing the test substance beneath a 4 ply, 2.5 x 2.5 cm surgical gauze patch. Each gauze patch was placed laterally from the midline of the back on the left front quadrant of the exposure area and secured with a strip of non-irritating adhesive tape. A strip of clear polyethylene film was placed over the patch and securely taped.
- Exposure period: each animal was placed in a restrainer for approximately six hours. At the end of the exposure period, the animals were removed from the restrainers, the wrappings and patches were removed, and the animals were returned to their cages.
- Frequency of applications: animals were treated once weekly for three weeks. Induction treatments were on Days 1, 8 and 15.
- Concentrations: 400 mg of test substance moistened with 0.5 ml of com oil.
- Control group: control group (5 animals) remained untreated during the induction phase of the study.

B. CHALLENGE EXPOSURE
After a two-week rest period, all animals (treated and untreated) were each challenged at a virgin test site.
- Concentrations: 400 mg of test substance moistened with 0.5 ml of com oil.
- Day of challenge: on day 29
- Application: as for the induction treatment, animals were treated by introducing the test substance beneath a 4 ply, 2.5 x 2.5 cm surgical gauze patch. Each gauze patch was placed laterally from the midline of the back on the right front quadrant of the exposure area and secured with a strip of non-irritating adhesive tape. A strip of clear polyethylene film was placed over the patch and securely taped.

OBSERVATIONS and SCORING METHOD
Observations for skin reactions at each test site were made approximately 24 hours after each treatment. In addition, observations for skin reactions were made approximately 48 hours after the first induction treatment and 48 hours after the challenge treatment.

An average score for each time period was obtained by adding all of the scores for each time period and dividing by the number of test sites scored for that tie period. The test substance is considered a sensitizer if the mean irritation scores, the total number of animals with scores, and/or the total number of scores for the virgin test site ion the test group after the challenge treatment are appreciably greater than those for the naive challenge group.
The Buehler sensitisation scoring scale was used, based on the erythema observed:
no reaction: score 0
Very faint, usually nonconfluent: score 0.5
Faint, usually confluent: score 1
Moderate: score 2
Strong, with or without oedema: 3
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene (DNCB). Conc. administered: induction 0.4 ml of 1 % w/v solution in 80 % ethanol; challenge 0.4 ml of 0.1 % w/v solution in acetone

Results and discussion

Any other information on results incl. tables

The test substance produced no irritation in the naive control animals after the single treatment at challenge.

The test substance likewise produced no irritation in the test animals after the challenge treatment and therefore did not elicit a sensitizing reaction in guinea pigs.

Group Mean challenge scores
Neive control 0.0
Test group 0.0

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU
Conclusions:
Not sensitising
Executive summary:

A skin sensitization study was conducted on 15 male and 15 female short-haired albino guinea pigs to determine if test substance produced a sensitizing reaction. Males and females were assigned to each of two goups, designated Groups I (5/sex) and II (10/sex). Group I animals remained untreated during the induction phase of the study and served as a negative control group. Group II animals, the test group, were treated with 400 mg of test substance moistened with 0.5 ml of corn oil (selected from previous screening). The animals were treated once weekly for three weeks, for a total of three treatments. After a two week rest period, all animals (Groups I and II) were challenged at a virgin test site with an application of 400 mg of test substance moistened with 0.5 ml of corn oil. The test substance produced no irritation in the naive control animals (Group I) after the single treatment at challenge. The test substance likewise produced no irritation in the test animals (Group II) after the challenge and therefore did not elicit a sensitizing reaction in guinea pigs.

Conlusion

Not sensitising