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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:
06 Dec 2001-27 Feb 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesanstalt für Pflanzenbau und Pflanzenschutz
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
study was conducted before existence of validated LLNA-method

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-phenoxyethanol (Protectol PE)
- Physical state: Liquid, colorless, clear
- Analytical purity: Purity 99.9% active substance
- Lot/batch No.: 66-4287
- Stability under test conditions: The stability of the test substance for the study period was determined by analysis.
- Storage condition of test material: Room temperature, exclusion of oxygen (storage under N2)

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Hsd Poc: DH (SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, FRG
- Age at study initiation: about 7 weeks
- Weight at study initiation: 349 g - 398 g
- Housing: stainless-steel wire mesh cages with plastic-coated grating, minimum floor area: 2,000 cm2; 5 animals per cage
- Diet (e.g. ad libitum): Kliba Labordiät (Kaninchen! Meerschweinchen-Haltungsdiät) Provimi Kliba SA, Kaiseraugst, Switzerland
- Water (e.g. ad libitum): tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
olive oil
Concentration / amount:
0.1 mL of 0.1 % test substance in vehicle
Day(s)/duration:
one week
Adequacy of induction:
other: causing mild to moderate irritation
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
1 mL undiluted test substance
Day(s)/duration:
48 h
Challenge
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
0.5 mL undiluted test substance
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10
Details on study design:
RANGE FINDING TESTS:
Four animals received intradermal injections of test substance (5%, 1% and 0.1% in olive oil Ph./DAB or in 1:1 Freund's adjuvant in 0.9% saline). Reactions were assessed after 24 and 48 hours. The 5% and 1% test substance preparations led to necrotic skin lesions. It was decided to use the locally and systematically well-tolerated 0.1% test substance preparation in the main study.
In the epicutaneous pretest, the test substance was applied undiluted and as 25, 50, and 75% test substance solution in Lutrol E 400 to the skin of five animals for 24 hours under occlusive dressing. Readings were performed 1 h, 24 h and 48 h after patch removal. Since no visible skin reaction was seen with the undiluted test substance at 24 h and 48 h after patch removal it was decided to use undiluted test substance for the main test.

MAIN STUDY
A. INDUCTION EXPOSURE
Ten animals received three pairs of 0.1 ml intradermal injections in the neck region (saline FCA alone, 0.1% test substance in olive oil Ph./DAB, and 0.1% test substance in saline FCA). Control animals were treated with saline FCA, olive oil Ph./DAB, or a 50% formulation of olive oil Ph./DAB in saline FCA; no test substance was added.
Epicutaneous induction was carried out one week later in the same region by applying 1 ml undiluted test substance to the skin, under occlusive dressing for 48 hrs.

B. CHALLENGE EXPOSURE
For challenge 0.5 ml undiluted test substance was applied to the flank of each animal of the test group and the second control group; the animals of the first control group remained untreated. Challenge was performed two weeks after epicutaneous induction under occlusive conditions, for 24 hours.

Examination:
Body weight was recorded at beginning and end of the study. Animals were observed for signs of toxicity twice a day and daily on weekends and holidays. Readings of skin reactions were made 24 hrs after intradermal induction, 48 hrs after start of epicutaneous induction, and at 24 and 48 hrs after removal of the challenge patch.
Evaluation:
Skin reactions were assessed according to the grading scale of Magnusson and Kligman.
Positive control substance(s):
yes
Remarks:
cinnamic acid (85 % historical control)

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.1%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.1%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Mortalities, clinical signs: Body weight gain was as expected; no mortalities or other signs of toxicity were seen.

After the intradermal induction intense erythema and swelling were observed at the injection sites of all control group animals and all test group animals to which only Freund's adjuvant/0.9% aqueous NaCl-solution (1:1) was applied. At the injection sites of a 0.1% test substance preparation in Freund's adjuvant/0.9% aqueous NaCl-solution (1:1) intense erythema and swelling were seen in all test group animals.

Injections of a 0.1% test substance preparation in olive oi1 Ph. Eur./DAB caused moderate and confluent erythema and swelling in all test group animals.

The control group animals, injected with olive oil Ph. Eur./DAB showed moderate and confluent erythema and swelling. A 50% formulation of olive oil Ph. Eur./DAB with Freund's adjuvant/0.9% aqueous NaCl-solution (1:1) caused intense erythema and swelling in all control group animals.

The epicutaneous induction with the undiluted test substance led to incrustation, partially open (caused by the intradermal induction) in addition to moderate and confluent erythema and swelling in all test group animals.

The challenge with the undiluted test substance did not cause any skin reactions neither in control group 1 nor in the test group 24 and 48 hours after removal of the patches.

9/10 animals treated with the positive control substance were sensitised.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met