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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:
September 2018 - January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
Substanceclass is know to lead to false positive results in LLNA Test, therefore a Guineapig is conducted.

Test material

Constituent 1
Reference substance name:
Sulfuric acid, mono-C8-18-alkyl esters, magnesium salts, compds. with triethanolamine
EC Number:
287-840-3
EC Name:
Sulfuric acid, mono-C8-18-alkyl esters, magnesium salts, compds. with triethanolamine
Cas Number:
85586-38-5
IUPAC Name:
85586-38-5
Test material form:
solid
Remarks:
dried from of a reaction mixture, water and cyclohexanol where evaporated
Details on test material:
Homogeneity: The test item was homogeneous by visual inspection.
Storage stability: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Expiry date: February 2020
Storage conditions: Room temperature
pH-value: Approx. 7 (moistened with water, determined by Bioassay Laboratories)
Specific details on test material used for the study:
For anayltical details see study report No. 18L00152

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
Dunkin Hartley, Crl:HA, SPF
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: spf
- Age at study initiation: no specified
- Weight at study initiation: 250 g – 274 g
- Housing:

Type of cage:
Stainless steel wire mesh cages with grating with shallow cage body; floor area: 4225 cm2
Enrichment:
Wooden gnawing blocks (Type KNH E-041); Abedd ® Lab. and Vet. Service GmbH Vienna, Austria
Bedding:
H 15005-29; Ssniff, Spezialdiäten GmbH (Experimental Animal Diets Inc., 59494 Soest, Germany)
Number of animals per cage:
5

- Diet (e.g. ad libitum): FD1 SQC; SDS Special Diets Services, 67122 Altrip, Germany, ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period:Acclimatization period of at least 5 days before the beginning of the experimental phase


ENVIRONMENTAL CONDITIONS
- Temperature (°C):20 +- 3°C;
- Humidity (%):30 - 70%
- Air changes (per hr):10
- Photoperiod (hrs dark / hrs light):12h/12h

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400
Concentration / amount:
1st induction (60 %): suspension (paste)
2nd and 3rd induction (50%): suspension (paste)
Day(s)/duration:
Exposure period: 6 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Concentration / amount:
Concentration test item 50 % (w/w)
Day(s)/duration:
Duration of exposure: 6 hours
Readings: 24, 48 and 72 h after the removal of the patch
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20
Details on study design:
RANGE FINDING TESTS:
In accordance with the cited guidelines a mild irritating concentration should be used in the main test for induction, whereas the maximum non-irritant concentration should be applied for challenge.
Amount applied: 0.5 g or 0.5 mL of the test item preparation was applied to each animal.
Occlusive dressing: 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test item preparations were applied to the skin of the flanks under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (4 x 4 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
Duration of exposure: 6 hours
Application site: Right and left flank anterior and posterior
Application frequency: One application
Number of test animals: 3
Readings: 1, 24, 48 and 72 h after removal of the patch
The animals were distributed as follows:
Flank region
(application site) Concentration % Number of Animals per Group
Left anterior 60 3
Left posterior 50 3
Right anterior 25 3
Right posterior 10 3
In the pretest, the highest concentration that could be technically used was 60% (w/w).

MAIN STUDY
Number of animals of the control group:10
Number of animals of the test group:20
A. INDUCTION EXPOSURE
3 inductions were conducted (day 0, 7 and 14)
Amount applied: 0.5 g of the test item preparation were applied to each test group animal. The control group received 0.5 mL of the vehicle.
Occlusive dressing: 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test item preparation or vehicle were applied to the skin of the flank under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (4 x 4 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
Duration of exposure: 6 hours
Application site: Intact flank
Test item preparation (test group): left flank anterior
Vehicle (control group): left flank anterior
Due to distinct skin irritation in the test group the patches were shifted to the middle part of the left flank during the third induction.
Concentration of test item: 1st induction: 60 % (w/w)
2nd and 3rd induction: 50 % (w/w)
Application frequency: Three times at weekly intervals; days 0, 7 and 14 on the same or shifted application area
Readings: 24 h after the removal of the patch
The control group was treated analogously to the test group but only with the vehicle without the test item.



B. CHALLENGE EXPOSURE
The challenge was carried out 13 days after the third induction.
Amount applied: 0.5 g of the test item preparation and 0.5 mL of the vehicle were applied to each animal.
Occlusive dressing: 2 x 2 cm gauze patches (6 layers surgical gauze Ph. Eur. from Lohmann GmbH & Co. KG) containing the test item preparation or vehicle were applied to the skin of the flank under an occlusive dressing. The dressing consisted of rubberized linen patches (4 x 4 cm from Russka), patches of Idealbinde (4 x 4 cm from Pfälzische Verbandstoff-Fabrik) and Fixomull® Stretch (adhesive fleece) from Beiersdorf AG.
Duration of exposure: 6 hours
Application site: Intact flank
Test item preparation: right flank posterior
Vehicle: left flank posterior
Concentration test item: 50 % (w/w)
Readings: 24, 48 and 72 h after the removal of the patch
The control group was treated analogously to the test group.

Positive control
A positive control (reliability check) with a known sensitizer is not included in this study. However, a separate study is performed twice a year in the laboratory.
The positive controls with Alpha-Hexylcinnamaldehyde, techn. 85% showed that the test system was able to detect sensitizing compounds under the laboratory conditions chosen.
Positive control substance(s):
yes
Remarks:
A positive control (reliability check) with a known sensitizer is not included in this study. However, a separate study is performed twice a year in the laboratory.

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: Total
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
other: In total, 4/20 test group animals showed skin reactions after 24 and 48 hours after removal of the patches.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
none
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
none

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Executive summary:

"Confidential item" was tested for its sensitizing effect on the skin of the guinea pig in the BUEHLER Test based on the method of BUEHLER, E.V. (1965) and according to OECD Guideline 406 (1992), the council regulation (EC) No 440/2008 part B.6. (2008) and the U.S. EPA OPPTS 870.2600 (2003) and MAFF 8147, 2000 guideline.
The test item concentrations for the main test were selected based on the results of the pretest, in which 3 guinea pigs of the same animal strain in total were used. In the pretest, the highest concentration that could be technically used was 60% (w/w). Therefore, descending concentrations of 60, 50, 25 and 10% (w/w) in polyethylene glycol 400 were chosen.
Based on the data of the pretest, the first induction was performed with a 60% (w/w) test item preparation in polyethylene glycol 400. Due to distinct local skin findings after the first induction, the second and third induction was performed with a 50% (w/w) test item preparation in polyethylene glycol 400. Likewise, a 50% test item preparation in polyethylene glycol 400 was chosen for the challenge application.
The main study was performed using 1 control group, which consisted of 10 animals and 1 test group, which consisted of 20 animals.
The inductions were performed on days 0, 7 and 14.
A challenge was carried out 13 days after the last induction.
In the main study, the animals did not show any signs of systemic toxicity.
As expected, no local skin findings could be observed in the control group neither after the inductions nor the challenge.
In the test group, 13 animals (13/20) revealed discrete or moderate erythema (grade 1 or 2) after the first induction, while after the second induction these skin findings were observed in 18 animals (18/20) animals. 13 animals (13/20) showed discrete or moderate erythema after the third induction.
After the challenge 4 animals (4/20) in total showed the following skin findings:
 Discrete erythema (grade 1)


Due to the unambiguous findings of the challenge, a second challenge was not performed.
Based on the results of this study and applying the evaluation criteria cited in chapter 3.8, it was concluded that "confidential testitem" has a sensitizing effect on the skin of the guinea pig in the BUEHLER Test under the test conditions chosen.

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