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Toxicological information

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:
From May 20, 2009 to November 5, 2009
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: MAFF in Japan, 12-Nousan-No. 8147, 2-1-6
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
GPMT test conducted in 2009 prior to worldwide acceptance of the LLNA.

Test material

Constituent 1
Chemical structure
Reference substance name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Cas Number:
915972-17-7
Molecular formula:
C33H39NO9
IUPAC Name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Test material form:
solid
Details on test material:
- Analytical purity: >94 %
Specific details on test material used for the study:
Lot/Batch no.: 08072
Description: Pale yellow green powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Oh-hara production facility (Shizuoka, Japan) of Japan SLC, Inc.
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: ca. 7 weeks at dosing
- Weight at study initiation: 371 – 479g
- Housing: Groups of 5 animals housed in cages (485 mm in width x 570 mm in depth x 360 mm in height)
- Diet: Certified pellet diet LRC4 (Lot No. 090305; Oriental Yeast Co., Ltd., Tokyo, Japan)
- Water: Tap water, ad libitum
- Acclimation period: 11 days before application

ENVIRONMENTAL CONDITIONS
- Temperature: 21.8 – 22.4°C
- Humidity: 21.8 – 22.4°C
- Air changes (per hr): 10 or more
- Photoperiod: Alternating 12-hour artificial light and dark cycles. light on at 7:00 a.m. and off at 7:00 p.m.).
- IN-LIFE DATES: From 02-Jun-2009 to 26-Jun-2009

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Remarks:
or a 1:1 w/w mixture of Freunds’ Complete Adjuvant with water
Concentration / amount:
1% (intradermal) and 50% (epicutaneous occlusive)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Remarks:
or a 1:1 w/w mixture of Freunds’ Complete Adjuvant with water
Concentration / amount:
50%
Adequacy of challenge:
other: Maximum practical concentration that could be prepared and dosed topically and did not give rise to irritating effects.
No. of animals per dose:
20
Details on study design:
RANGE FINDING TESTS: Yes
Intradermal injections: In 2 guinea pigs, 0.5 and 1%, w/v. test substance was injected; skin responses were assessed approximately 24 hour later
Topical applications: 4 guinea pigs (2 concentrations / animal), 5, 10, 25 and 50%, w/w; reaction sites were assessed for erythema and edema at 24 hours after removal of the patch

Result of range finding tests:
Intradermal injections: Diffuse or patchy erythema (score 1) and/or moderate dispersed erythema (score 2) were observed on the 0.5% and 1% injected skin sites of 2 animals.
Topical applications: No visible change (score 0) were found on the 5 and 50% of the test substance applied skin sites of 2 animals, and on the 10 and 25% of the test substance applied skin sites of 2 animals.

MAIN STUDY

The following 4 groups were set:
Group/ Induction Exposure / Challenge Exposure
A/ Test item/ Test item
B/ None/ Test item
C/ HCA/ HCA
D/ None/ HCA
HCA: α-Hexylcinnamaldehyde (positive control)

A. INDUCTION EXPOSURE

A.1. Intradermal induction (first induction)
- Test group: 1 (A) (20 animals)
- Control groups: 3 (B, C, D) (10/ 10/ 5 animals)
- Site: Shoulder region of each animal was clipped using an electric clipper
- Frequency of applications: 3 pairs of intradermal injections (0.1 mL each)
- Duration: 7 days
- Concentrations: 1%
[1] Emulsion of FCA (Freund’s complete adjuvant) and SPS (sterilized physiological salt solution) at the rate of 1:1 (v/v)
[2] Suspension of the test substance or solution of HCA in liquid paraffin
[3] Emulsion of the test substance in FCA blended with SPS at the rate of 1:1 (v/v) or emulsion of HCA in FCA blended with SPS at the rate of 1:1 (v/v)
The animals in Group B and D were given similar injections without the supplementation of the test substance and HCA.

A.2. Topical induction (Reinduction)
- Exposure period: On Day 7 after the intradermal induction
Pretreated with 0.5 mL of 10% sodium lauryl sulfate (Kanto Chemical Co., Inc.) in white petrolatum by rubbing on Day 6 after the intradermal induction (after clipping on the day before the topical induction).
- Test group: 1 (A) (20 animals)
- Control groups: 3 (B, C, D) (10/ 10/ 5 animals)
- Site: On the day before application, the shoulder region of each animal was clipped again.
- Frequency of applications: Once
- Duration: 48 hours
- Concentrations: 50%
Group A, 0.4 g of the test substance formulation
Group C, 0.4 mL of the HCA formulation
Animals in the Group B and D were treated with the corresponding vehicle used for preparation of the test substance and HCA formulation.

B. CHALLENGE EXPOSURE
- Day(s) of challenge: On Day 14 after the topical induction
- Exposure period: 24 hours
- Test group: 1 (A) (20 animals)
- Control groups: 3 (B, C, D) (10/ 10/ 5 animals)
- Site: 2 cm x 2 cm of the left flank for each animal. In addition, the white petrolatum for Groups A and B or liquid paraffin for Groups C and D was treated on the right flank of each animal.
- Concentrations: 0.2 g of the test substance formulation (50%) in white petrolatum for Groups A and B or 0.2 mL of HCA formulation (10 %) in liquid paraffin for Groups C and D was applied
- Evaluation (hr after challenge): At 24 and 48 hours after the challenge patch removal

Challenge controls:
The positive control with alpha-hexylcinnamaldehyde techn. 97.8% showed that the test system was able to detect sensitizing compounds under the laboratory conditions chosen.
Positive control substance(s):
yes
Remarks:
HCA: α-Hexylcinnamaldehyde (positive control)

Results and discussion

Positive control results:
Group C:
In the HCA-treated group, 8 of 10 animals exhibited the reaction of score 2 (moderate dispersed erythema), 1 animal exhibited the reaction of score 1 and the remaining 1 animal exhibited the reaction of score 0.

Group D:
On the other hand, all 5 animals in the unsensitized / HCA-challenged group exhibited score 0.
Therefore, the sensitization rate of HCA-treated group was calculated to be 90% (9/10) and the skin sensitization was evaluated to be Classification V (extreme). This was considered to sufficiently assure the reliability of this study.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met