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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:
09 June 1992 - 03 July 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study. This well documented study is performed according to OECD and EC guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

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)
Version / remarks:
1984
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The skin sensitisation test was performed in 1993 prior to first publication of the LLNA method by OECD in 2002.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diphenyl in Pastillen
- Substance type: light yellow, crystalline
- Physical state: solid
- Analytical purity: 99,9% based on analytical substance balance (GC/normalisation, taking into account water and sublimation)
- Impurities (identity and concentrations): 0,01% sublimation residues and 0,09% unknown compounds
- Purity test date: start: 24-01-1992, end: 16-03-1992
- Lot/batch No.: 372745/1992
- Expiration date of the lot/batch: 06-01-1994
- Stability under test conditions: analytically determined: substance is minimal 30 minutes stable in the test solution
- Storage condition of test material: in absence of light in a refrigerator
- Supplier: Bayer AG
- Other: pH 5.5 (saturated aqueous solution)

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Bor:DHPW
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen/Kreis Paderborn, Germany
- Age at study initiation: 4 to 7 weeks
- Weight at study initiation: average: 317 g (range: 286 - 352 g)
- Housing: during acclimation period and experimental time: Makrolon cages type IV, 5 animals per cage; the cages were changed every 2 days for cages with clean litter. Low-dust wood granules were used as bedding material (Ssniff Spezialdiäten, GmbH, Soest). The wood granules were randomly tested for contaminant levels. The records of the investigation are kept at BAYER AG. The analysis results showed no evidence of any impact on the study objectives
- Diet (e.g. ad libitum): Altromin 3020 - maintenance diet for guinea pigs (Altromin GmbH, Lage), pellets with 3 mm diameter - ad libitum
- Water (e.g. ad libitum): Tap water ad-libitum with drinking water quality; during the experiment two 750 ml polycarbonate bottles were foreseen per cage.
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1,5°C
- Humidity (%): 40 - 70
- Air changes (per hr): ca. 12 - 15
- Photoperiod (hrs dark / hrs light): 12 hours (artificial light from 6 until 18h00)

- Healthy symptom-free animals
- No vaccination or treatment of animals with anti-infective substances were performed before delivery of the animals, during the acclimation or during treatment
- Females were nulliparous and non-pregnant

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Concentration / amount:
Intradermal induction: 5%
Topical induction: 40%
Challenge: 40%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
Intradermal induction: 5%
Topical induction: 40%
Challenge: 40%
No. of animals per dose:
20 animals in the treated group
2 control groups with 10 animals (2nd control in case no clear results after first challenge are obtained or in case of a positive result after the first challenge in order to study concentration dependency)
Details on study design:
RANGE FINDING TESTS:
- intradermal induction: 1 guinea pig: 2 times injection of 0,1 mL of following concentrations of diphenyl: 0%; 1%, 2,5%; 5% (animal number 1). After 24 and 48 hours the injection areas are checked:
0%: grey area with red border, diameter ca. 0,6 cm
1%: white area with red border, diameter ca. 0,4 cm
2,5 - 5%: white area with red border, diameter ca. 1 cm
- topical induction: 4 guinea pigs exposed to 4 concentrations of the test substance (6%, 12%, 25% and 50%). The test substance was impregnated on a plaster (4 plasters per animal) and applied under occlusive conditions for 24 hours. After the exposure the substance is washed away with physiological saline and the skin is shaved near by the area of exposure. Skin reactions are scored 48 and 72 hours after the start of the application. Scores for skin erythema and edema were 0 for all test animals and all concentrations tested at 48 and 72 hours.
- challenge exposure: one week before the challenge exposure, 5 guinea pigs are tested to determine the challenge concentration. Each animal received 4 with test substance impregnated plasters under occlusive conditions for 24 hours. At the end of the exposure, the substance is removed with physiological saline and the skin in the surrounding area is shaved. Skin reactions were scored 48 and 72 hours after the start of the application. No symptoms of erythema neither of edema were observed at all concentrations in all animals at 48 and 72 hours.
Remark: precipitation was observed in the range-finding test for topical induction at the 50% concentration. It was therefore advised to stirr the test solution during 30 minutes (with a magnetical stirrer). As the objective of 50% solution with stirring was not reached, it was decided to test 40% test solution in the main topical induction and challenge exposure.

MAIN STUDY
A. INDUCTION EXPOSURE (intradermal induction)
One day before application the back and the flanks of the guinea pigs are shaved. Behind the necks at left and right side from the spinal column, three injections on one line are given to the guinea pigs. The distance between the injections was 1-2 cm; the application volume was 0,1 mL. The treatment of the test animals was performed as follows:
1) cranial injection: FCA (Difco Lab.) 1:1 mixture with physiological saline (sterile)
2) medial injection: 5% diphenyl in 'Pastillen' mixed with polyethyleneglycol 400
3) caudal injection: 5% diphenyl in 'Pastillen' mixed with polyethyleneglycol 400 and FCA in similar proportion

Control animals: control animals are treated under similar conditions as test animals, however injections mentioned above under 2) and 3) do not contain diphenyl but an appropriate quantity of polyethyleneglycol 400

B. INDUCTION EXPOSURE (topical induction)
The topical induction was performed 1 week after the intradermal induction. One day before the treatment areas were shaved and a 10% sodium lauryl sulphate solution in paraffin oil was applied. Between the injection sites, hypoallergic plasters (2 x 4 cm) are applied, covered with aluminium foil and fixed to the skin with an adhesive tape. The plasters were treated as follows:
a) test group: 0,5 mL diphenyl in 'Pastillen' 40%
b) control group: 0,5 mL polyethyleneglycol 400
At the end of the 48 hours exposure the substance is removed with physiological saline.

C. CHALLENGE EXPOSURE
The challenge is performed 3 weeks after the intradermal induction. One day before the challenge, the backs and flanks of the animals are shaved. The animals of the test group and the first control group are treated with a 40% test solution impregnated hypoallergic plaster at the left side for 24 hours which is fixed to the skin with self adhesive tape. At the right side, which serves as a control, only a plaster impregnated with polyethyleneglycol is applied and fixed. The application volume is 0.5 mL. At the end of the exposure, the substance is removed with sterile physiological saline and the skin is shaved around the treatment area.
Challenge controls:
At the right side, which serves as a control, only a plaster impregnated with polyethyleneglycol is applied and fixed. The application volume is 0.5 mL. At the end of the exposure, the substance is removed with sterile physiological saline and the skin is shaved around the treatment area.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamic aldehyde, 2-mercaptobenzothiazole, 4-aminobenzoic acid ethyl ester

Results and discussion

Positive control results:
Sensitivity and reliability of the GPMT test method (according to Magnusson and Kligman) is ensured and tested regularly. The appropriate documents are stored in the archives of the test lab.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
40%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No clinical symptoms observed
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 40%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No clinical symptoms observed.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
40%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No clinical symptoms observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 40%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No clinical symptoms observed.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
40%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical symptoms observed
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 40%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical symptoms observed.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
40%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical symptoms observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 40%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical symptoms observed.

Any other information on results incl. tables

Results of general examinations:

- No deaths were observed

- The changes in body weight of the treated animals corresponded to those of the control groups:

day of first application: mean body weight of control group one: 321 g; standard deviation: 22

day 24: mean body weight of control group one: 437 g; standard deviation: 30

day of first application: mean body weight of control group two: 317 g; standard deviation: 19

day 24: mean body weight of control group two: 434 g; standard deviation: 24

day of first application: mean body weight of the test substance group: 315 g; standard deviation: 14

day 24: mean body weight of the test substance group: 437 g; standard deviation: 23

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Diphenyl did not have skin sensitisation potential under the conditions of this maximization test in guinea pigs.