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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:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Reference substance name:
2,4-di-tert-butylphenyl diphenyl phosphite; 2-tert-butylphenyl diphenyl phosphite; 4-tert-butylphenyl diphenyl phosphite; triphenyl phosphite
EC Number:
941-802-9
Molecular formula:
Unspecified
IUPAC Name:
2,4-di-tert-butylphenyl diphenyl phosphite; 2-tert-butylphenyl diphenyl phosphite; 4-tert-butylphenyl diphenyl phosphite; triphenyl phosphite
Details on test material:
- Physical state: yellowish liquid
- Storage condition of test material: In the original container, at room temperature (range of 20 ± 3 °C), away from direct sunlight.
- Stability under test conditions: Stable in polyethylene glycol for at least 1 hour

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Wolferstrasse 4, CH-4414 Fullinsdorf / Switzerland
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: Pretest groups: 322 - 416 g
- Housing: Individually in Makrolon type-4 cages with standard softwood bedding
- Diet: Pelleted standard Provimi Kliba 3418, batch nos. 90/01 and 91/01, guinea pig breeding / maintenance diet, containing Vitamin C (Provimi Kliba AG, CH-4303 Kaiseraugst), ad libitum
- Water: Community tap water, ad libitum
- Acclimation period: One week for the control and test group under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr):10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Concentration / amount:
intradermal induction: 100%
epidermal induction: 100%
epidermal challenge: 3%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG 300
Concentration / amount:
intradermal induction: 100%
epidermal induction: 100%
epidermal challenge: 3%
No. of animals per dose:
15 males (main study); 5 males (pretest)
Details on study design:
RANGE FINDING TESTS:
INTRADERMAL INJECTIONS
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of A = 100 %, B = 75 % and C = 50 % of the test item. PEG 300 was used for the dilutions. The three concentrations were determined during non-
GLP formulation trials performed prior to the pretest. Dermal reactions were assessed 24 hours later. Based on the results, the test item concentration of 100 % was selected for intradermal induction in the main study.

EPIDERMAL APPLICATIONS
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (3x3 cm) were saturated with the test item at D = 100 %, E = 75 %, F = 50 % and G = 25 % and applied to the clipped and shaved flanks. PEG 300 was used for the dilutions. The volume of test item preparation applied was approximately 0.2 ml. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test item. The dressings were removed after an exposure period of 24 hours. Twenty-one hours after removal of the dressing the application site was depilated with an approved depilatory cream in order to visualize any resulting erythema. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman. No highest non-irritating concentration could be determined after the epidermal pretest described above. Therefore, a second pretest was performed with two additional naive guinea pigs (nos. 286 - 287), treated in the same way as those described previously, with the concentrations of H = 15 %, I = 10 %, J = 5 % and K = 3 % in PEG 300, using an application volume of approximately 0.2 ml. Based on the results obtained the concentration selected for induction and challenge in the main study was 100 % and 3 %, respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test item, at 100%
3) The test item, at 100%
Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) PEG 300
3) 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
One week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of filter paper was saturated with the undiluted test item and placed over the injection sites of the test animals. The volume of test item preparation applied was approximately 0.3 ml. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test item. The guinea pigs of the control group were treated as described above with PEG 300 only, also applied at a volume of approximately 0.3 ml. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

B. CHALLENGE EXPOSURE
The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way. Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (3x3 cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 3 % (applied to the left flank) and the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and vehicle applied was approximately 0.2 ml. The dressings were left in place for 24 hours. Twenty-one hours after removal of the dressing the test sites treated with the test item were
depilated as described in the epidermal pretest. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
3%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 3%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
3%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 3%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
3%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 3%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
3%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 3%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

There were no deaths during the course of the main study, hence no necropsies were performed.

No signs of systemic toxicity were observed in the animals.

Animal no. 274 of the control group showed a loss of body weight (2.8 %) during the acclimatization period. It recovered between the treatment start and the end of the study. The body weight of the other animals was within the range commonly recorded for animals

of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the findings in an adjuvant sensitization test (M&K-test) in guinea pigs and in accordance to Commission Directive 96/54/EEC, te test item does not have to be classified and labelled as a skin sensitizer.
Executive summary:

In order to assess the cutaneous allergenic potential of the test article, the Maximization-Test was performed in 15 (10 test and 5 control) male albino guinea pigs, in accordance with OECD Guideline No. 406 and the Directive 96/54/EEC, B.6. The intradermal induction of sensitization in the test group was performed in the nuchal region with the undiluted test item and an emulsion of Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the undiluted test item one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 3 % in PEG 300 and PEG 300 alone under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing. None of the control and test animals showed skin reactions after the challenge treatment with the test article at 3 % (w/w) in PEG 300. Based on the above mentioned findings in an adjuvant sensitization test (M&K-test) in guinea pigs and in accordance to Commission Directive 96/54/EEC, te test item does not have to be classified and labelled as a skin sensitizer.