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Administrative data

Description of key information

Study conducted to recognised training guidelines with GLP certification.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Mar 8, 1999 to May 18, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
When this study was performed, the LLNA did not yet exist.
Species:
guinea pig
Strain:
other: Albino (Ibm:GOHI; SPF quality guinea pigs, synonym: Himalayan spotted)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC LTD. Biotechnology & Animal Breeding Division, Wölferstrasse 4, CH-4414 Füllinsdorf, Switzerland)
- Age at study initiation: 4-6 weeks
- Weight at study initiation: 290-345 g
- Housing: Individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132, Muttenz)
- Diet (e.g. ad libitum): Pelleted standard Nafag Ecosan 845 25W4, Batchs nos. 103/98, 112/98 and 23/99), guinea pig breeding/maintenance diet ("Nafag", Nähr- und Futtermittel AG, CH-9202 Gossau) ad libitum
- Water (e.g. ad libitum): Community tap water from Füllinsdorf ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-28
- Humidity (%): 32-84
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 (Fluorescent "Gold" lamps (Sivania Gold F40T1260))


IN-LIFE DATES: From: March 8, 1999 To: May 18, 1999
Route:
intradermal and epicutaneous
Vehicle:
other: FCA/saline 1:1 (intradermal)
Concentration / amount:
0% test material
0.3 ml
Day(s)/duration:
Day 1
Adequacy of induction:
not specified
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Concentration / amount:
5% test material
0.3 ml
Day(s)/duration:
Day 1
Adequacy of induction:
not specified
Route:
intradermal and epicutaneous
Vehicle:
other: FCA/saline 1:1 (intradermal)
Concentration / amount:
5 % test material
0.3 ml
Day(s)/duration:
Day 1
Adequacy of induction:
not specified
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
25% test material
0.2 ml
Day(s)/duration:
Day 22 (48 hours)
Adequacy of challenge:
not specified
No. of animals per dose:
Control: 5
Treatment: 10
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 5, 3 and 1 % of the test article in PEG 400.
Dermal reactions were assessed 24 hours later. All three tested concentrations resulted in dermal reactions with a score of 2.

Based on the results and the provisions of OECD guideline 406, a test article concentration of 5 % 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 article at A = 100 %, B = 75 %, C = 50 % and D = 25 % and applied to the clipped and shaved flanks. PEG 400 was used for the dilutions. The volume of test article 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 article. The dressings were removed after an exposure period of 24 hours.

Approximately 21 hours after removal of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) in order to visualize any resulting erythema.
The depilatory cream was placed on the patch sites euid surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.
The reaction sites were assessed approximately 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

Based on the results obtained the concentration selected for induction and challenge in the main study was 100 % and 25 %, respectively (see table 1)

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 4
- Exposure period: 3 weeks

-First induction period - Test groups: 3 pairs of intradermal injections were made at the border of a 4X6 cm area in the hair-free clipped region a follows:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, at 5 % in PEG 400.
3) The test article at 5 % in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
- Control group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) PEG 400
3) 1:1 (w/w) mixture of PEG 400 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
-Second induction period
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 article and placed over the injection sites of the test animals. The volume of test article 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 ensures intensive contact of the test article.
The guinea pigs of the control group were treated as described above with PEG 400 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
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24 hours
- Test groups: 25% (same method as second induction period)
- Control group: PEG (same method as second induction period)
- Volume applied: 0.2 mL
- Site: dorsal
- Evaluation (hr after challenge): 24 and 48h after removal of the bandage
Challenge controls:
The animals of the control group were intradermally induced with PEG 400 and FCA/physiological saline and epidermally induced with PEG 400 under occlusion.
Positive control substance(s):
yes
Remarks:
2-MERCAPTOBENZOTHIAZOLE
Positive control results:
In this study 90 % (at the 24-hour reading) and 100 % (at the 48-hour reading) of the animals of the test group were observed with very slight to moderate/severe erythematous reactions after treatment with a non-irritant 2-MERCAPTOBENZOTHIAZOLE concentration of 10 % in mineral oil.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Draize scores for erythema and edema were 0 in all animals.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Draize scores for erythema and edema were 0 in all animals..
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Draize scores for erythema and edema were 0 in all animals.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Draize scores for erythema and edema were 0 in all animals..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Draize scores for erythema and edema were 0 in all animals.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Draize scores for erythema and edema were 0 in all animals..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Draize scores for erythema and edema were 0 in all animals.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Draize scores for erythema and edema were 0 in all animals..
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10%
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
The animals of the test group were observed with very slight to moderate/severe erythematous reactions after treatment with a non-irritant 2-MERCAPTOBENZOTHIAZOLE concentration of 10 % in mineral oil
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
The animals of the test group were observed with very slight to moderate/severe erythematous reactions after treatment with a non-irritant 2-MERCAPTOBENZOTHIAZOLE concentration of 10 % in mineral oil

Skin effects after intradermal induction:

- Control and test groups:

The expected and common findings were observed after the different applications using FCA intradermally and consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

Skin effects after epidermal induction:

- Control group:

No erythematous or oedematous reaction was observed in the animals treated with PEG 400 only.

- Test group:

Discrete/patchy erythema was observed in 5 (at the 24 -hour reading) and 6 (at the 48 -hour reading) out of 10 animals after treatment with the undiluted test article.

Skin effects after the challenge:

- Control and test groups:

No skin reactions were observed in the animals when treated with either PEG 400 only or when treated with the test article at 25% in PEG 400.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance applied at a challenge concentration of 25% in PEG 400 does not cause skin sensitization in the guinea pig maximization test.
Executive summary:

In this guideline (OECD 406) study conducted with GLP certification, the test material (EC 231-272-0) did not induce skin sensitisation in the test animals (Guinea pig). The test was conducted using the Guinea pig maximisation method, with an induction of 5% test material applied for 48 hours exposure under occlusive conditions, one week after induction injections. A challenge exposure of 25% test material was applied under occlusive conditions, 24 hour exposure, two weeks after the induction. Test animals showed no signs of sensitisation to the test material during the challenge and observation periods. Based upon the results of the test, the test material does not meet the criteria for classification under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The substance has been tested for skin sensitization in three studies of which only one study is suitable for hazard evaluation. The oldest study (Merck 1980) was performed with five animals per group and a non-standard testing design (Guinea pig percutaneous skin sensitization assay by application with Q-tips). The test substance was applied in pure form for induction and as a 10% solution in peanut oil for challenge. Dinitrochlorobenzene was used as positive control. Whereas dinitrochlorobenzene resulted in skin reactions in all five animals, no skin reactions were observed for the test or vehicle group animals. The study has obvious limitations such as the low number of animals and the deviations from the OECD testing guidelines.

A later study (Merck 1985) is a maximization test according to Magnusson. It was performed under GLP, but differs in reporting and performance requirements from the requirements of OECD guideline 406. Most importantly, no results of a pre-test are listed and no test substance challenge of vehicle-induced animals was performed. It is therefore not possible to decide whether the skin reactions observed upon challenge with 50% in liquid paraffine are due to skin irritation or to skin sensitization. This is particularly important since in the later study (RCC 1999), the pre-experiment revealed skin irritation after treatment with 50% in PEG400. Therefore, this study cannot be used for hazard assessment and the results are unreliable (Klimisch 3). For further information, 2% were used for intradermal induction and 50% for epidermal induction.

 

The most recent study (RCC 1999) was performed following OECD testing guideline 406 and GLP. It fulfills all validity criteria (Klimisch 1).A skin maximisation test in guinea-pig was performed with ten test and five control guinea-pigs. The purity of the test material was 99.6%. Concentrations for induction and epidermal challenge were chosen based on the outcome of range-finding experiments. Intradermal induction caused skin reactions at dose levels of 1, 3 and 5% and therefore 5% which is the highest recommended concentration was chosen for the main study. The vehicle was polyethylene glycol (PEG 400) with Freund's Complete Adjuvant (FCA)/ physiological saline.

Epidermal induction was performed with undiluted material. The pre-test had shown that skin reactions were noted after treatment with 50, 75 and 100%, but not after treatment with 25%. Therefore, 25% was chosen as the challenge concentration.

During the epidermal induction period, seven of the ten animals showed skin reactions after treatment. After the epidermal challenge with the test substance, none of the animals showed skin reactions. In contrast, 90% of the animals were sensitized in the positive control group using 2-MERCAPTOBENZOTHIAZOLE as positive control substance. The substance was found to be a non-sensitizer in this study.

The substance does not contain structural alerts for protein binding as screened with the OECD QSAR toolbox v3.3 including the skin metabolism simulator.


Migrated from Short description of key information:
To evaluate the allergic potential of the test substance, a skin maximisation test (OECD 406, GLP) in ten test and five control guinea-pig was performed. The test substance did not induce dermal reactions in test animals 24h and 48h after challenge, respectively. Thus, the test substance is considered to be as a non-sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.