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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:
2008-05-14 to 2008-08-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
July 17th, 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid GPMT conducted according to guideline is available, which is reliable without restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. Moreover, no indication for skin sensitisation was observed in this study, thus, no dose response information is needed. For this reason and for reasons of animal welfare no additional LLNA was conducted.

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kissleg, Germany
- Age at study initiation: approximately 4 weeks
- Weight at study initiation: 360.6 ± 22.8 9 (test group) and 355.7 ± 15.4 9 (control group) at start of the main experiment
- Housing: with 2 or 3 animals in Makrolon-cages No. IV
- Diet: ad libitum, type "3023" from Altromin International, Lage, Gerrnany as pelleted diet (Batch No.: 0638; expiry date: 2008-11-19)
- Water: ad libitum, tap water
- Acclimation period: 14 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 -70
- Air changes (per hr): 8 times/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: sesame oil
Concentration / amount:
Intradermal: 0.5 % (w/w) solution of the test substance in sesame oil
Day(s)/duration:
day 0
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
Dermal: 75 % (w/w) solution of the test substance in sesame oil
Day(s)/duration:
day 7
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: sesame oil
Concentration / amount:
15 % (w/w) solution in sesame oil
Day(s)/duration:
day 21
No. of animals per dose:
19 (test group 10, control group 5, pilot experiment 4)
Details on study design:
RANGE FINDING TESTS:
Intradermal: 0.1 ml of 5 %, 3.5 %, 2 % and 0.5 % (w/w) solution in sesame oil.
Dermal: soaked patch with 100 % and 75 % (w/w) solution in sesame oil.
Challenge: soaked patch with Duhring chamber with 50 %, 25 %, 15 % and 5 % (w/w) in sesame oil

-- Intradermal application:
- 1 animal was shorn on the right and left flank over an area of approximately 4 cm x 6 cm.
- Animals received 0.1 ml of various concentrations by disposable syringe.
- Observation: after 24 h

-- Dermal application:
- Soaked patches of 2 cm x 2 cm size with various concentrations of the test substance in sesame oil were applied to the right and left flank.
- Dressing: The patches were covered with Blenderm (3 M company, St. Paul, USA) and bandaged on with Acrylastic (Beiersdorf AG, Hamburg, Germany) which was fixed by Leukoplast (Beiersdorf AG, Hamburg, Germany).
- Observation: after 24 h

-- Challenge application:
- Duhring chambers (diameter 4 cm) were filled with a soaked patch of 2 cm x 2 cm size with various concentrations of the test substance in sesame oil and applied to the skin. 24 hours before the flanks of the animals had been shorn.
- Dressing: The gauze patch and the Duhring chamber were applied each to one flank of a guinea pig, covered with
Micrapore (3 M company, St. Paul, USA) and bandaged on with Acrylastic (Beiersdorf AG, Hamburg, Germany) which was fixed by Leukoplast (Beiersdorf AG, Hamburg, Germany).
- Observation: after 24 hours

MAIN STUDY
Based on the results of the pilot experiment the following concentrations were chosen for the main experiment:
Intradermal: 0.1 ml of 0.5 % (w/w) solution of the test substance in sesame oil.
Dermal: soaked patch with 75 % (w/w) solution of the test substance in sesame oil.
Challenge: soaked patch with Duhring chamber with 15 % (w/w) solution in sesame oil

A. INTRADERMAL INDUCTION (day 0)
- Exposure period: 24 h
- Test groups:
(1) 0.1 ml per side FCA (mixed at a ratio of 1:1 in vehicle)
(2) 0.1 ml per side of the 0.5 % (w/w) solution of the test substance found to be slightly irritant in the pilot experiment.
(3) 0.1 ml per side of the 0.5 % (w/w) solution of the test substance found to be slightly irritant in the pilot experiment in FCA (mixed at a ratio of 1:1 in vehicle).
- Control group: treated similarly - with the exception that they received only the diluting vehicle agent
- Site: on the back over an area of 4 cm x 6 cm below the shoulder blade
- Concentrations: 0.5 % (w/w)

B. DERMAL INDUCTION (day 7)
- Exposure period: 48 h
- Site: same part of the skin was dermally treated
- A soaked patch (2 cm x 4 cm) with the 75 % (w/w) solution of the test substance was placed over the injection area and fixed in the way described in the pilot experiment.
- Concentration: 75 % (w/w)
C. CHALLENGE EXPOSURE (day 21)
- Exposure period: 24 h
- Site: Areas of 5 cm x 5 cm on the flanks of the animals. Left flank: test substance, right flank: control with vehicle
- Duhring chambers (0 4 cm) were filled with soaked patches (2 cm x 2 cm) with the 15 % (w/w) solution of the test substance in sesame oil
- Evaluation (hr after challenge): 24 h, 48 h (After 24 hours the plaster was removed. After a further 21 hours after removal of the plaster the skin was shorn. Three hours later the skin reaction was assessed. This assessment was repeated after a further 24 hours.)
- Concentration: 15 % (w/w)

SCORING SYSTEM
erythema and scurf formation:
- no reddening: 0
- slight reddening (difficult to detect): 1
- easily detectable (erythema): 2
- medium (erythema): 3
- strong (erythema) (dark red) to slight formation of scurf (damage deep down): 4
edema formation:
- no swelling: 0
- slight swelling (hardly detectable): 1
- easily recognizable swelling (clearly marked by elevation): 2
- medium edema (swelling of approx. 1 mm): 3
- strong edema (swelling over 1 mm and clearly stretching beyond the treated area): 4
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole 98 %, Sigma-Aldrich, Germany, Batch No. 04718CC-264 in sesame oil

Results and discussion

Positive control results:
A validation experiment was performed according to the method of Magnusson and Kligman with 2-Mercaptobenzothiazole 98 % in sesame oil and produced a sensitisation rate of 90 % showing the sensitivity of the test system. (2008-02-25 to 2008-03-28)

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
15% (w/w)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
15 % (w/w)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
15 % (w/w)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
15 % (w/w)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
2-Mercaptobenzothiazole 98%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
2-Mercaptobenzothiazole 98%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Pilot experiment:

- The intradermal application of the 5 %, 3.5 % and 2 % (w/w) test substance in sesame oil showed medium reactions, but the 0.5 % (w/w) solution of the test substance in sesame oil elicited a slight reaction.

- Dermal application without Duhring Chambers of 100 % and 75 % (w/w) solutions of the test substance in sesame oil showed slight reactions. Dermal application of 50 % and 25 % (w/w) test substance in sesame oil with Duhring Chambers provoked a medium or slight reaction, respectively, whereas the 15 % and 5 % (w/w) test substance in sesame oil with Duhring Chambers showed no reaction.

- Control group: no reactions

Main study

Induction:

- The intradermal and the epicutaneous induction resulted in slight skin reactions in a various number of animals indicating that the chosen test substance concentrations led to slight irritation. In the control group animals no skin reaction was observed after the application of the vehicle sesame oil.

- Additional observations: Crust formation (diameter: 6 mm) was observed at the injection sites with FCA in the test group and the control group animals 1-2 weeks following the intradermal injection.

Challenge:

- No visible changes of the treated skin sites were observed in the test group animals 24 h and 48 h after patch removal (= grading "0").

- In the control group animals also no visible signs of skin reactions were observed (= grading "0") indicating that the chosen concentration of the test substance was a subirritative dose.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study the test substance is not a dermal sensitizer.
Executive summary:

In a dermal sensitization study according to OECD guideline 406 with Isostearic acid, esters with methyl α-D-glucoside (100% UVCB substance), Dunkin-Hartley guinea pigs were tested using the method of Magnusson and Kligman. Positive control substance was 2-Mercaptobenzothiazole.

Based on the results of the pilot study, for the intradermal and epicutaneous induction procedure test substance concentrations of 0.5 % and 75 % in sesame oil were used, respectively. The test article concentration for the challenge procedure was 15 % in sesame oil.

Slight skin reactions were observed after induction, whereas at challenge no visible changes of the treated skin sites (no erythema and no edema) were observed in test or control animals at any time point. 

In this study Isostearic acid, esters with methyl α-D-glucoside is not a dermal sensitizer.