Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 April 2007 to 23 May 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study was already available

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Reactive Yellow 176 Ester
Reactive Yellow 176 Sulfato

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Species and strain: Guinea pigs, Dunkin Hartley
Source: LAB-ÁLL Bt. Budapest, 1174 Hunyadi u. 7.
Justification of the strain: The guinea pig is the standard species for skin sensitisation study.
Sex: Female
Weight range at the beginning of the study: 300-346 g
Acclimatization time: 14 days
Date of animal receipt: 12 April 2007
Animal health: Only animals in acceptable health condition were used for the test. It is certified by the veterinarian.
Cage type: Animals were housed in macrolon cages, size III., with 3 or 2 animals/cage (42 x 42 x 19 cm)
Bedding: Laboratory bedding, SSNIFF Lignocel 3-4 Fasern
Produced by: SSNIFF Specialdiaten GmbH, Ferd.-Gabriel-weg 16, 59494 Soest
Animal room: 602/8
Light: 12 hours daily from 6 a.m. to 6 p.m. (artificial light)
Temperature: 20 ± 3 °C
Relative humidity: 30-70 %
Food and Feeding: Animals received PURINA Base – Lap gr. diet for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi road, Hungary, ad libitum.
Water Supply: The animals received tap water, as for human consumption, ad libitum containing 50 mg/100 ml Ascorbic acid. The drinking water is routinely analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Identification: The animals were marked individually with ear punching. The cages were marked with individual identity cards with information about study code, sex, cage number, dose group and individual animal number.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
5% / 0.1 mL
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
50% / 0.5 mL
50% was the highest technically apllicable concentration
Day(s)/duration:
Day 8 for 48 h
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
50% / 0.5 mL
Day(s)/duration:
Day 22 for 24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Range finding study: 2 animals/two concentrations (in one animal two concentrations were tested: one concentration in the left side, and other concentration in the right side).

Main study:
Test groups: 10 animals
Control group: 5 animals
Details on study design:
Control animals were treated similarly to test animals, except that during the induction phase, the test item was omitted.
Induction involved two main procedures: intra-dermal treatments (Main Study I) and dermal exposure (Main Study II) with closed patch technique. The intra-dermal and the dermal induction treatments were observed and recorded. Records are archived with the raw data.

Main Study I: Intra-dermal Induction Exposure
Before starting the exposure an area of approximately 5 x 5 cm on the scapular region of animals was clipped free of hair and shaven close with care.

Intra-dermal treatment
Test groups:
A row of three injections, six in all, was made on each side of test animals, as follows:
2 injections with 0.10 ml of Freund's Adjuvant mixed with physiological saline (1:1 v/v),
2 injections with 0.10 ml of the test item (5 %) homogenized in physiological saline,
2 injections with 0.10 ml of test item (5 %), formulated in a 1:1 (v/v) mixture of
Freund's Adjuvant and methyl cellulose (1 %),

Control group:
The control animals were treated similarly as the test group however, the vehicle, without the test item was used for injections, as follows:
2 injections with 0.1 ml mix of Freund's Adjuvant and physiological saline (1:1)(v/v),
2 injections with 0.1 ml of physiological saline,
2 injections with 0.1 ml of 50 w/v% physiological saline, in a 1:1 mixture (v/v) Freund's Adjuvant and physiological saline.

Main study II: Dermal Induction Exposure
Six days after the intra-dermal injections, in all animals the test area was painted with 0.5 ml of 10 % sodium dodecyl sulphate in Vaseline 24 h prior to topical induction application, in order to create a local irritation.
Approximately 24 hours after the painting, the test animals were exposed to test item on the other hand the control animals were treated with physiological saline, as vehicle.
Closed patch was applied in the following manner: in case of the test animals 0.5 ml of test item (at concentration of 50 %) was spread on the surface prepared previously and covered with a standard (5x5 cm) size of porous gauze patch.
Control animals were treated dermally with 0.50 ml of physiological saline, as vehicle and the dressing was prepared and applied as for the test animals.
The exposed areas were covered for 48 hours with porous gauze fastened with "Leucoplast" (Closed Patch Test).

The dermal irritation scores (in case of the preliminary study (primary irritation) and in cases of induction dermal exposures) were evaluated according to the scoring system by Draize.
Challenge controls:
Main study III: Challenge Exposure
Two weeks after the dermal treatment the animals were exposed to the challenge dose, dermally. 24 hours before the challenge treatment the left and the right flank areas (5x5 cm) of each animal were prepared for application. The challenge was performed as a dermal exposure (Closed Patch Test).
Left shaved flank areas of the animals (both the test and the control) were treated with 0.5 ml of the test item (at concentration of 50 %). The right shaved flank areas were treated with 0.5 ml of physiological saline, in all cases. Implementation was done according to prescriptions. Time of exposure was 24 hours.
Positive control substance(s):
yes
Remarks:
2-MERCAPTOBENZOTHIAZOLE,

Results and discussion

Positive control results:
In the test group 10 animals were treated with the reference item.
After the challenge with the reference item 2-MERCAPTOBENZOTHIAZOLE, positive response was seen in six out of ten animals in the test group (60 %). The mean of the scores were 0.6 and 0.4 according to the 24th and 48th-hour results. The dermal scores represented discrete erythema developed on the skin of sensitised guinea pigs. On the opposite (right) side treated with vehicle no reaction was found.
Five control animals were exposed to a vehicle during induction treatment and they were treated with the reference item on the challenge day only.
No visible changes were found at the 24th and 48th hour examinations. During the challenge exposure, the reference item 2-MERCAPTOBENZOTHIAZOLE (at concentration of 25 % (w/v)) did not evoke primary irritation.

On the basis of the results of the present reliability study, the reference item 2-MERCAPTOBENZOTHIAZOLE evoked positive response in 60 % of the test animals.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Intra-dermal induction exposure: 0.1% (w/v) Dermal induction treatment: 75% (w/v) Challenge treatment: 25% (w/v)
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Intra-dermal induction exposure: 0.1% (w/v) Dermal treatment: 75% (w/v) Challenge treatment: 25% (w/v)
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

(MAIN STUDY III)

DERMAL RESPONSE SCORES FOR GUINEA PIGS CHALLENGED WITH THE TEST ITEM (CONTROL AND TEST GROUP)

(Challenge concentrations: 50%)

 

TEST ANIMALS

CONTROL ANIMALS

ANIMAL NUMBER

SCORES OF DERMAL REACTION

ANIMAL NUMBER

SCORES OF DERMAL REACTION

24 hours

48 hours

24 hours

28 hours

After the patch removal

After the patch removal

253

0

0

251

0

0

255

0

0

252

0

0

256

0

0

254

0

0

257

0

0

263

0

0

258

0

0

264

0

0

259

0

0

-

-

-

260

0

0

-

-

-

261

0

0

-

-

-

262

0

0

-

-

-

265

0

0

-

-

-

Mean of scores

0.00

0.00

Mean of scores

0.00

0.00

Number of positive/number of tested

0/10

0/10

Number of positive/number of tested

0/5

0/5

Scoring of Skin Sensitisation

0 = no visible change

1 = discrete of patchy erythema

2 = moderate and confluent erythema

3 = intense erythema and swelling

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Challenge with test item Gelb Sulfato evoked no positive responses in the test animals sensitised previously. At the same time, none of the animals proved to be positive in the control group. The net response value represented an incidence rate of 0 % and the net score value of 0.00.

According to the net percentage value of positively responded animals and to the net score value of the skin reactions, the test item Gelb Sulfato was classified as a non-sensitiser.
Executive summary:

A skin sensitisation study was performed according to Magnusson-Kligman method using Freund's complete adjuvant technique to evaluate the sensitisation potential of test item Gelb Sulfato.

 

The study was performed in accordance with the study plan, the OECD Guidelines for Testing of Chemicals No. 406, Directive 96/54 EEC B.6 and the Principles of Good Laboratory Practice (GLP) and reported with a GLP certificate.

 

10 test animals were subjected to sensitisation procedures in a two-stage operation, i.e. an intra-dermal treatment and a topical application. The test item was used at concentration of 5 % for intra-dermal injections and at concentration of 50 % for dermal sensitisation treatment. Before the dermal exposure the test area was painted with 0.5 ml of 10 % sodium dodecyl sulphate in Vaseline 24 h prior to the topical induction application, in order to create a local irritation. Two weeks following the last induction exposure, a challenge dose (at concentration of 50 %) was administered.

 

Challenge was performed by dermal application of the test item.

 

5 control guinea pigs were simultaneously exposed to vehicle during the sensitisation phase and they were treated with the test item (at concentration of 50 %) only in the case of challenge.

Incidence Rate 

No signs of contact sensitisation were detected in guinea pigs exposed previously to the test item during experiments.

 

Intensity of Sensitisation Response

In the control and treated animals the mean of the scores was 0.00 according to the 24th and 48th-hour results.