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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:
July - August 2001
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted version 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed in 2001. The Test guideline (TG) for the determination of skin sensitization in the mouse, the LLNA (TG 429) was adopted in 2002.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
446-620-9
EC Name:
-
Cas Number:
120983-72-4
Molecular formula:
Hill formula: C5H6Cl2O CAS formula: C5H6Cl20
IUPAC Name:
2-Chloro-1-(1-chlorocyclopropyl)ethanone
Test material form:
liquid
Details on test material:
purity: 92.3 %
Specific details on test material used for the study:
Purity 92%
The stability of the test item in the vehicle was analytically verified for up to 2 hours.

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Hsd/Win:DH
Sex:
male

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
intradermal induction: 0.1%
topical induction: 3% ;

Test item group
1. Injection site: cranial/bilateral complete Freund's adjuvant (DIFCO LAB.) diluted 1:1 with sterile physicological saline solution
2. Injection site: medial/bilateral 0.1% JAU 6476 Chloromethylketone formulated in corn oil
3. Injection site: caudal/bilateral 0.1% JAU 6476 Chloromethylketone formulated at equal parts in sterile physiological saline solution and complete Freund's adjuvant.
Day(s)/duration:
2d (48h)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
corn oil
Concentration / amount:
0.5% (2.5 mg test substance/animal)
Day(s)/duration:
1d (24h)
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
test group: 20 animals
control group: 10 animals
Details on study design:
Intradermal Induction: The dorsal region and the flanks of the guinea pigs were shorn one day prior to the application. Starting behind the nape of the neck, three injections each in a row were made on the left and the right side of the spinal column. The 1st and 2nd injections were made as contiguous as possible and the 3rd injections in a distance of about 2 cm from the 2nd. The volume applied per injection site was 0.1 ml.
The topical induction was performed one week after the intradermal induction. On the day prior to topical treatment, the test areas of the animals were shorn. Hypoallergenic patches (2x4 cm) were placed between and on the injection sites, covered with aluminum foil and held securely in place on the skin using a ORABAND® self-adhesive tape (Fa. Orafol). At the end of the 48-hour exposure period, the remaining test item was removed with sterile physicological saline solution and the treatment areas were visually assessed.
The challenge was performed three weeks after the intradermal induction. In the meantime, the test item concentrations for the challenge had been determined in a dose range-finding study using 2 guinea pigs that were treated during the inductions in the same manner as the control animals (see page 26). The dorsal region and the right flank of the animals were shorn one day prior to the challenge. During the challenge a hypoallergenic patch loaded with the 0.5% test item formulation was placed on the right flank (caudal) of the animals of the test item group and the control group and held securely in place on the skin with a ORABAND® self-adhesive tape for 24 hours. A patch loaded only with the vehicle was placed also on the right flank (cranial) as control. The volume applied in each case was 0.5 ml. At the end of the exposure period, the remaining test item was removed with physiological saline solution, and 21 hours later the skin of the animals was shorn in the zone of the challenge area.
Positive control substance(s):
no
Remarks:
alpha-Hexylzimtaldehyd used in a separate test for reliability of the method

Results and discussion

Positive control results:
Reliability of the Method:
The Guinea Pig Maximization Test methodology was checked for reliability in a test on female guinea pigs using alpha-Hexylzimtaldehyd formulated in sterile physicological saline solution at the concentrations indicated below:
For the intradermal induction a 5% test item formulation was used, and for the topical induction a 25% formulation. After the challenge with a 12% test item formulation 100% of the test animals exhibited dermal reactions in the challenge treatment. There was no reddening of the skin to be observed on control group animals. The sensitivity as well as the reliability of the experimental technique is thus confirmed by this study.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
skin reactions grade 3
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
skin reactions grade 1 - 3
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
red wheals and encrustations after induction, no skin reactions after challenge
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
red wheals and encrustations after induction, no skin reactions after challenge
Remarks on result:
no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
skin-sensitization potential; hazard class H317 ; 100% of animals reacted positively after challenge with grade 1-3
Executive summary:

The Guinea Pig Maximization Test (GPMT) was performed in 2001 on 30 female guinea pigs (20 animals for the test item group and 10 control animals) to determine whether the test item JAU 6476 Chloromethylketone (purity: 92.0%) exhibits skin sensitizing properties. Additional two animals were used for dose-finding. The study was conducted according to OECD Guideline No. 406/EC Guideline 96/54/EC (22th Adaptation of Guideline 67/548/EEC)/Health Effects Test Guideline, OPPTS 870.2600 with the following test item concentrations:


Intradermal induction: 0.1%


Topical induction: 3%


Challenge: 0.5%


The test item was formulated in corn oil to yield a solution.


The challenge with the 0.5% test item formulation led to skin effects (grade 1 - 3) in 20 of 20 animals (100%) in the test item group and no skin effects were seen in the control group animals. In summary, under the conditions of the maximization test and with respect to the evaluation criteria the test item therefore exhibits a skin-sensitization potential.

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