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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:
Study initiation: November 25, 2011; Final report: February 29, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Remarks:
Korea
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Previously existing study, conducted in Korea

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Chemical Name : ESA (1,3,2-Dioxathiolane,2,2-dioxide)
Received date : 22 November 2011
Appearance : pale-yellow solid
Purity : 99.1 %
Storage condition : Refrigerate ( 2~8 ºC)
Supplier : LG chemical Ltd

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: SPF/VAF Crl:IAF (HA)-hr(Hairless)
Sex:
male
Details on test animals and environmental conditions:
Producer: ORIENT BIO INC. (Address; 143-1, Sangdaewondong, Jungwon-gu, Seongnam-si, Gyeonggi-do, Korea)
Supplier: ORIENT BIO INC. (Address; 143-1, Sangdaewondong, Jungwon-gu, Seongnam-si, Gyeonggi-do, Korea)
Reason for choice of the Species: Guinea pig have been applied widely in skin sensitisation tests as a suitable experimental animal for toxicity testing. In addition, sufficient raw data has been accumulated and is available for interpretation and evaluation of study results.
Date of acquisition : 02 December 2011
Number of animals received : 40 male animals
Body weight on arrival : 279.75 g - 328.93 g
Quarantine and acclimation: Quarantine was carried out by examining animals' appearance at the day of receiving. Animals were acclimated more than 5 days from receiving day. (Only animals with good appearance were selected for the study after observation during the acclimation period)
Body weight at the Administration : 393.06 g - 425.37 g
Number of used animals : 6 animals for pilot test, 30 animals for main test
Grouping: Animals were weighed one day before the test substance administration, and grouped to ensure a distribution of graded body weight by ExcelTM over 300g.
Identification of animals: Individual animals were identified by skin marking with an picric acid during acclimation, and ear punching after acclimation using the individual card labeling.
The record sheets provided at the entrance of the SPF animal room contained the study number, the study title, the duration of the SPF room use, the name of the study director and the name of study personnel.
Disposal of remaining animals : Remains and study end animals were euthanized by CO2.

Animal care facility
Temperature and humidity during test work: 22.5 ± 1.0 ºC and 46.9 ± 5.1 %RH
Ventilation frequency: 10 - 15 air changes / hr
Lighting cycle: 12 hours duration (lighting on at 8 a.m. and off at 8 p.m.)
Lighting intensity: 281 Lux.
Ambient noise level: 53.4 dB
Ammonia concentration: less than 5 ppm
Housing: All animals were housed in a plastic cage (810 W × 610 L × 260 H mm) during acclimation, administration and observation period. During the experiment, not more than 5 animals were housed in each cage.
Feed: Laboratory guinea pig feed, Purina Korea, Co. Ltd. (Address; 627, Jangdang-dong, Pyeongtaek-si, Gyeonggi-do, Korea). Supplied ad libitum. Analysis carried out by supplier, there was no factor that may affect the study.
Water: Incheon, Korea municipal tap water purified by reverse osmosis filtering system. Supplied ad libitum (Including Vitaimn C, Peterpet INC.). Analysis was performed by national certificated inspection organization. And, there was no factor that may affect the study.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
12.5 % (0.1 ml uised for intradermal injection and 0.5 ml applied dermally with an occlusive patch)
Day(s)/duration:
Single injection intradermally. Occlusive patch was applied for 48 hours.
Adequacy of induction:
other: Test concentration used in pilot study did not cause irritation, but skin pre-treated with 10% SDS.
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
12.5 % (0.5ml)
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 animals treated with test substance and 10 control animals
Details on study design:
Determination of concentration
The concentration of test substance was determined from a pilot study and used for induction and challenge exposure.

Determination of induction dose level (Intradermal injection ; I.D., pilot test)
Test substance of each concentration with FCA were injected 0.1 ml in I.D.. And two animals were used in each concentration. The highest concentration not to cause skin necrosis was selected for the concentration of test substance used for I.D. induction. (Concentration of pilot study : 12.5 %, 10 %, 7.5 %, 5 %)
As a result of pilot test, all animals were not shown skin necrosis. Therefore, highest concentration(12.5 %) was determined as I.D. induction dose.

Determination of induction and challenge dose levels (Occulsive patch, pilot test)
Test substance of each concentration applied 0.5 ml to the test area and attached by an occlusive patch for 24 hours. Two animals were used in each concentration. The concentration of test substance used for induction exposure was the lowest concentration that cause erythema in skin. And the concentration of test substance used for challenge exposure was the highest non-irritant concentration. (Concentration of pilot study : 12.5 %, 7.5 %)
As a result of pilot test, all animals were not shown skin irritation. Therefore, highest concentration(12.5 %) was determined as induction and challenge concentration.

Reason for selecting concentration
Pilot tests were carried out and doses were chosen by the Good Laboratory Practice standards and the Test Guidelines of the National Institute of Environment Research (NIER) [Notice No. 2010-29, (Revised 16th August, 2010)], and OECD Guideline for Testing of Chemicals No. 406 'skin sensitisation' (Adopted 17th July, 1992).

Method of administration
1) Intradermal injection (1st Induction, Day 0)
Three pairs of I.D. of 0.1 ml volume were given in the shoulder region which was cleared of hair so that one of each pair lies on each side of the midline.

Test substance:
Up: 1:1 mixture of FCA and physiological saline
Middle: Test substance (12.5 %)
Low: Test substance (12.5 %) formulated in an 1:1 mixture of FCA and physiological saline

Negative control:
Up: 1:1 mixture of FCA and physiological saline
Middle: Distilled water
Low: 50% w/v formulation of the vehicle in an 1:1 mixture of FCA and physiological saline.

2) The test area was painted with 0.5 ml of 10 % sodium dodecyl sulfate at 24 hours prior to 2nd induction.

3) Occulsive patch (2nd Induction, Day 7)
Test substance: A cotton pad (4 cm2) was fully-loaded with 0.5 ml test substance (12.5 %) and applied to the test area and held in contact by an occlusive patch for 48 hours.
Negative control: The vehicle only was applied in a similar manner to the test area (4 cm2) and held in contact by an occlusive patch for 48 hours.

4) Challenge (Day 21)
12 days after inductions had been completed, challenge exposure was performed in all animals. The flanks of treated and control animals were cleared of hair. A cotton pad (4 cm2) patch loaded with 0.5 ml test substance (12.5 %) was applied to left flank of the animal. The patches were held in contact by an occlusive patch for 24 hours.

Administration time : Morning of the administration day

General observations

Clinical signs and mortality:
General clinical signs or mortality of all treated animals were observed continuously during the experimental periods once every day up to the final challenge observation.

Body weight:
Individual animals were weighed at receiving, grouping, 1st induction, 2nd induction and 24, 48 hours after removal of the challenge patch.

Observations - treated and control groups:
Approximately 21 hours after rremoval of the challenge patch, Test area was cleaned and closely-clipped, and 3 hours later the skin reaction was observed and recorded according to the grades shown below.
Approximately 24 hours after this observation a second observation was made and once again recorded.

Determination of skin sensitisation
Skin reactions were evaluated according to the Test Guidelines of the National Institute of Environment Research (NIER) [Notice No. 2010-29, (Revised 16th August, 2010)], and OECD Guideline for Testing of Chemicals No. 406 'Skin sensitisation' (Adopted 17th July, 1992).
Challenge controls:
A cotton pad patch loaded with the vehicle only also was applied to right flank.
Positive control substance(s):
not specified
Remarks:
Study states that a positive control is conducted every 6 months but no details or results are provided

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
12.5 %
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
none observed
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
12.5 %
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
none observed
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
12.5 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none observed
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
12.5 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none observed
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
other: report states that a positive control is carried out every 6 months but does not give more detail
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
other: report states that a positive control is carried out every 6 months but does not give more detail

Any other information on results incl. tables

Clinical signs and mortality

All tested animals showed no significant clinical signs and mortality during the experimental period.

Body weight changes

All tested animals showed no significant body weight changes concerned with test substance during the experimental period.

Observations of applied site

The test substance 24 hours after removal of challenge patch, 9 heads of animals showed discrete or patchy erythema, 7 heads of animals showed moderate and confluent erythema .

48 hours after removal of challenge patch, 9 heads of animals showed discrete or patchy erythema and 7 heads of animals showed moderate and confluent erythema .

Negative control

24 hours and 48 hours after removal of challenge patch, no animal showed any other skin sensitisation reaction.

Determination of skin sensitisation

Final sensitisation rate of test substance was 80 % at 24 hours and 48 hours.

Final sensitisation rate of negative control was 0 % at 24 hours and 48 hours.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
This study was performed to evaluate the skin sensitisation of ESA in 20 male guinea pigs. Induction and challenge were conducted at dose of 12.5 % (w/v), and then the skin reaction was evaluated at 24 and 48 hours after removing challenge patch.
In the results, there were no significant clinical signs, body weight changes and mortalities concerned with the test substance during the experimental period.
In the test substance group, 9 heads of animals showed discrete or patchy erythema, 7 heads of animals showed moderate and confluent erythema at 24 hours after removal of challenge patch. And 9 heads of animals showed discrete or patchy erythema and 7 heads of animals showed moderate and confluent erythema at 48 hours after removal of challenge patch.
In the negative control, there was no skin reaction at 24 and 48 hours after removal of challenge patch.
In conclusion, the sensistisation study in the SPF male Hartley guinea pig with ESA, test substance did not affect the clinical sign, mortality and body weight changes. And, final sensitisation rate of ESA was 80 % in this study.
Executive summary:

This study was performed to evaluate the skin sensitisation of ESA in 20 male guinea pigs. Induction and challenge were conducted at dose of 12.5 % (w/v), and then the skin reaction was evaluated at 24 & 48 hours after removal of challenge patch.

(1) All animals were not shown clinical signs and mortalities concerned with test substance during the experimental period.

(2) All animals were not shown body weight changes concerned with test substance during the experimental period.

(3) In treated group, sensitisation rate was determined as 80 % at 24 & 48 hours after challenge exposure.

Based on the results of the skin sensitisation study of ESA to male guinea pig, the sensitisation rate was determined as 80 %.