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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:
2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline, without GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This test was already available (it was performed for cosmetic regulation compliance), before REACH regulation requirements.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
DIPOTASSIUM GLYCYRRIHZINATE was used as the test substance.
This substance is a white to slight yellow powder with a characteristic sweet taste having a pH of 5.07, residue on ignition of 19.8%.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
Age : 4-week-old female Hartley guinea pigs
Body weight: 200-250 g
Origin : SHIMIZU Laboratory Supplies Co., Ltd. (37 Yoshida Shimo Adachi-cho, Sakyo-ku, Kyoto-shi)
Quarantined and acclimatized for 10 days.

During this period, general conditions were observed and body weights was measured. On the Iast day of the quarantine and acclimatization period, twenty healthy guinea pigs that had gained weight at a normal rate were selected for the study.

Temperature of 22 ±3°C
Humidity of 55 ±15%
Ventilation : all-fresh ventilation and illumination 12 hours per day (from 6 a.m. to 6 p.m.).

Each stainless bracket cage (350 mm (W) x 500 mm (D) x 420 mm (H) mm) housed two guinea pigs. The guinea pigs had free access to Solid Feed Lab R Stock for experimental animals (Nosan Corporation, 2-2-1 Minatomirai, Nishi-ku, Yokohama-shi, Kanagawa-ken) and tap water (Shinichi-cho water) placed in automatic water feeders. Each cage was tagged with a label indicating the study number, name of test substance, study group, and animal numbers. The guinea pigs were identified by writing an ID number on their coat using a picric acid saturated alcohol solution

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal
Vehicle:
water
Concentration / amount:
1.25%
Day(s)/duration:
7 days
Adequacy of induction:
other: minimal concentration of the substance exhibiting skn reactions
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
not specified
Concentration / amount:
0.2 mL of 10% substance
Day(s)/duration:
48h
Adequacy of challenge:
not specified
No. of animals per dose:
5 x 4 groups = 20 guinea pigs
Details on study design:
1) Sensitization
Hair on the back of the neck of every guinea pig was clipped using an electric clipper (0.05-mm blade, Daito Electric Machine IND. Co., Ltd.) to prepare a sensitization area of approximately 2 cm x 4 cm. Next, in each group, 0.05 mL each of the following agents 1), 2), and 3) were administered intracutaneously at three sites on. the left and right sides of the dorsal midline in a symmetrical mariner_
1) : The emulsion made of FCA and distilled water for injection
2) : Test substance solution
3) : The emulsion made of FCA and the test substance

2) Inflammatory agent application
On day 7 of intracutaneous sensitization, hair on the sensitization area was again clipped using an electrical clipper, and. 01 g of the 10% SLS ointment was then applied without occlusion to the guinea pigs in the test substance and negative-control groups.

3) Resensitization
At 24 hours after application of the inflammatory agent, lukewarm water was used to remove the inflammatory agent. In the test substance group, 0.2 mL of a 10% test substance resensitization solution was applied to surgical lint of 2 cm x 4 cm, and the lint was covered with an adhesive elastic bandage (SILKYTEX) and surgical tape for 48 hours. In the positive control group, the same procedures were performed using 0 2 mL of the 1% DNCB resensitization solution. In the negative control group, plain surgical lint cloth was covered in the saine marner for 48 hours (sham procedure).

4) Elicitation
At 18 days alter the resensitization, hair on the right ventral region of all guinea pigs was clipped using an electrical clipper to prepare an elicitation area of approximately 5 cm x 5 cm. In the test substance group, 0.1 mL of the 10% or 1% elicitation solution was applied to surgical lint of 2 cm x 2 cm, and. the lint was covered using an adhesive elastic bandage and surgical tape for 24 hours. In the positive control group, 0.1 mL of the 0.1% DNCB elicitation solution .was applied with occlusion in the same marrer. In the negative control group, the solutions used in the test and positive substance groups were applied with occlusion in the same marner.

5) Skin sensitization assessment
At 24 and 48 hours after the end of the elicitation procedure, the challenged areas were macroscopically examined and erythema and edema assessed in accordante with the criteria specified below.2) Furthermore, based on assessment scores, the average skin reaction scores were calculated to compare the severity of the skin sensitization resulting from the test substance with that of the negative con trol. Additionally, in all groups, the challenged areas mitre photographed during assessment.





Challenge controls:
0.2 mL of 1% DNCB for 48h (occlusive)

0.2 mL of 0% substance for 48h (occlusive)
Positive control substance(s):
yes
Remarks:
2,4-dinitrochlorobenzene (Lot No.: TWG2966, Wako Pure Chemical Industries, Ltd.; hereinafter referred to as "DNCB")

Results and discussion

Positive control results:
In the positive control group, erythema with scores of 2-4 points and edema with scores of 1-3 points were seen in the right ventral region of all guinea pigs after the 0.1% DNCB elicitation (the average scores 24 and 48 hours alter elicitation were 6.00 and. 5.60 points, respectively).

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
Total no. in group:
10
Clinical observations:
No
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
Total no. in group:
5
Clinical observations:
No
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
DNCB 0.1% soin
Total no. in group:
5
Clinical observations:
No
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
DNCB 1% soin
Total no. in group:
5
Clinical observations:
Erythema + Edema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
DNCB 0.1% soin
Total no. in group:
5
Clinical observations:
Erythema + Edema
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1.25% substance
Total no. in group:
5
Clinical observations:
No
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
Total no. in group:
5
Clinical observations:
No
Remarks on result:
no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Executive summary:

The skin sensitization study was performed with a OECD 406 similar guideline (maximisation in guinea pig). It was concluded that DIPOTASSIUM GLYCYRRHIZINATE is not a skin sensitizer