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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:
16 November 2011 to 23 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD, EU & US EPA test guidance in compliance with GLP and reported with a valid GLP certificate.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Reactive Red F03-0318

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: LAL/HA/BR
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Guinea pigs (LAL/HA/BR)
Source: LAB-ALL Bt. Budapest, 1174 Hunyadi u. 7.
Justification of strain: The guinea pig is the standard species used for skin sensitisation studies.
Number of animals:
Preliminary test: 4 females, 4 males
Main test: Test groups: 10 animals; Control group: 5 animals
Sex: Male
Body weight range at the beginning of the study: 240 - 329 g
Age of animals at arrival: Young adult, 8 weeks
Acclimatization time:6 days
Animal health: Only animals in acceptable health condition were used for the test as certified by the veterinarian.
Cage type: Animals were housed in macrolon cages, size III., with 2 or 3 animals/cage (42 x 42 x 19 cm).
Bedding: Laboratory bedding, Lignocel 3-4 Fasern (produced by J. Rettenmaier & Sohne GmbH+CO.KG, D-73494 Rosenberg, Germany) was available to animals during the study.
Animal room: 602/5
Light: 12 hours daily from 6 a.m. to 6 p.m. (artificial light)
Temperature during the main study: 20.0 - 24.0°C
Relative humidity during the main study: 25 -52 %
Ventilation:15-20 air exchange/hour
The environmental parameters were recorded twice daily during the study. Minor variations from the target temperature and relative humidity ranges were observed. These deviations were considered to have no impact on the animal health, as certified by the Clinical Veterinarian, or on the outcome of the study and interpretation of the results due to their low magnitude.

Food and Feeding: Animals received PURINA diet for rabbits (Batch number: 0570 08 11 Expiry date: 28 November 2011; Batch number: 0580 10 11 Expiry date: 16 January 2012; Batch number: 0050 11 11 Expiry date: 26 February 2012) produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi road, Hungary, ad libitum. This diet is classified as being suitable for Guinea pigs as the vitamin D level is high enough to meet the needs of this species. This is the diet used by the breeder/supplier and animals are fully adapted to this diet on arrival. The details of the diet or diets used will be archived with the raw data.
Water Supply: Animals received tap water from municipal supply as for human consumption, containing 50 mg/100 ml ascorbic acid, ad libitum. 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. Copies of the relevant Certificates of Analysis are retained in the archive at CiToxLAB Hungary Ltd.
Identification: The animals were individually marked using ear punching. The cages were marked with individual identity cards with information about study code, sex, cage number, dose group and individual animal number.
Randomisation: All animals were sorted according to weight on the day of the randomisation prior to the start of the treatment period. After this the animals were allocated to the test groups. The result of the randomisation was checked according to the actual body weights assuring an acceptable homogeneity and variability among the groups.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
Detailed in table form. See any other information on materials nad methods incl. tables.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
Detailed in table form. See any other information on materials nad methods incl. tables.
No. of animals per dose:
Detailed in table form. See any other information on materials nad methods incl. tables.
Details on study design:
Main Study: 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 results of 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
Approximately 24 hours before the treatment, an area of 5 x 5 cm on the scapular region of animals was clipped free of hair and shaved.

Intra-dermal treatment
Test groups:
A series of three injections was administered on each side of the scapular region of treatment group animals, as follows, resulting in six injections per animal:
2 injections with 0.10 ml of the test item in physiological saline at 5 % (w/v) concentration,
2 injections with 0.10 ml of test item in 5 % (w/v), formulated in a 1:1 (v/v) mixture of Freund's Adjuvant and physiological saline.

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 0.10 ml mix of Freund's Complete Adjuvant and physiological
saline (NaCl 0.9 %) (1:1) (v/v),
2 injections with 0.10 ml of physiological saline,
2 injections with 0.10 ml of a 1:1 (v/v) mixture of Freund's Adjuvant and physiological saline.
Main study II: Dermal Induction Exposure

The same inter-scapular region which received the intradermal injections, were used for dermal induction exposure. The test animals were treated with approximately 0.5 ml of the 50 % (w/v) concentration of the test item in distilled water. This was the highest possible dose; it was found non irritant on the skin of the guinea pigs in the preliminary dose range finding study. Control animals were treated with distilled water. The exposed areas were covered for 48 hours with 4 layers of porous gauze pads and fully occlusive foil fastened (Closed Patch Test). After the patch removal, any remaining test item was removed with a gauze swab.

Since the test item was not a skin irritant in the range-finding study, 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.

Following dermal induction treatments, the animals were left untreated for 14 days prior to challenge applications.

Main study III: Challenge Exposure
Two weeks after the topical induction application, the animals were exposed to a dermal challenge dose at the flanks. Twenty four hours before the treatment, the hair was removed from an area of approximately 6x8 cm on the left and right flank of each animal. A 5x5 cm2 patch of sterile gauze was saturated with the test item at 50 % (w/v) concentration in distilled water and applied to the left flank of all animals (both the test and the control group).
The right shaved flank area of all animals was treated with a 50 % dilution of the maximum dermal challenge dose (i.e. 25 (w/v) % in distilled water).
The volume of formulated test item and vehicle was approximately 0.5 ml. Treatment was as indicated in section 3.5.2.2 (Closed Patch Test). The time of the exposure was 24 hours. After the patch removal any remaining test item was removed with a gauze swab.
Challenge controls:
Main study III: Challenge Exposure
Two weeks after the topical induction application, the animals were exposed to a dermal challenge dose at the flanks. Twenty four hours before the treatment, the hair was removed from an area of approximately 6x8 cm on the left and right flank of each animal. A 5x5 cm2 patch of sterile gauze was saturated with the test item at 50 % (w/v) concentration in distilled water and applied to the left flank of all animals (both the test and the control group).
The right shaved flank area of all animals was treated with a 50 % dilution of the maximum dermal challenge dose (i.e. 25 (w/v) % in distilled water).
The volume of formulated test item and vehicle was approximately 0.5 ml. Treatment was as indicated in section 3.5.2.2 (Closed Patch Test). The time of the exposure was 24 hours. After the patch removal any remaining test item was removed with a gauze swab.
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole

Results and discussion

Positive control results:
Challenge with test item 2-Mercaptobenzothiazole resulted positive response in test animals sensitised previously. The net response values at the 24 and 48 hours observations represented an incidence rate of 70% and 50% and the net score values of 0.70 and 0.50 respectively. In the control animals no visible changes were found either at the 24 and 48 hours examinations or following challenge with the test item. The dermal scores represented discrete (score 1) erythema developed on the skin of sensitised guinea pigs.

On the basis of the results of the reliability check study, the test item 2-Mercaptobenzothiazole was classified as a skin sensitizer. This demonstrated that the experimental procedure was successful.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% (w/v)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50% (w/v). No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% (w/v)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% (w/v). No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% (w/v)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50% (w/v). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% (w/v)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50% (w/v). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
mean score 0.70
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 50%. No with. + reactions: 7.0. Total no. in groups: 10.0. Clinical observations: mean score 0.70.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
mean score 0.50
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 50%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: mean score 0.50.

Any other information on results incl. tables

Dermal Response Scores for Guinea Pigs Challenged With the Test Item (Challenge dose: 50% (w/v) in distilled water)

ANIMAL NUMBER

SCORES OF DERMAL REACTION

(Test animals)

ANIMAL NUMBER

SCORES OF DERMAL REACTION

(Control animals)

24 hours

48 hours

24 hours

48 hours

After the patch removal

After the patch removal

3

0

0

1

0

0

6

0

0

2

0

0

7

0

0

4

0

0

8

0

0

5

0

0

9

0

0

13

0

0

10

0

0

-

-

-

11

0

0

-

-

-

12

0

0

-

-

-

14

0

0

-

-

-

15

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

 Score of Skin Sensitisation

0 = no visible change

1 = discrete or patchy erythema

2 = moderate and confluent erythema

3 = intense erythema and swelling

 

Dermal Response Scores for Guinea Pigs Challenged with Reference Item 2-Mercaptobenzothiazole

Test Animal Number

Sex: Male

SCORES OF DERMAL REACTION

Control Animal Number

Sex: Male

SCORES OF DERMAL REACTION

24 hours

48 hours

24 hours

48 hours

After the patch removal

After the patch removal

34

1

1

38

0

0

36

0

0

39

0

0

37

1

0

42

0

0

40

1

1

45

0

0

41

1

0

46

0

0

43

0

0

-

-

-

44

1

1

-

-

-

49

0

0

-

-

-

50

1

1

-

-

-

51

1

1

-

-

-

Mean of scores

0.70

0.50

Mean of scores

0.00

0.00

Number of positive/number of tested

7/10

5/10

Number of positive/number of tested

0/5

0/5

Score of Skin Sensitisation

0 = no visible change

1 = discrete or patchy erythema

2 = moderate and confluent erythema

3 = intense erythema and swelling

 

OBSERVATIONS RECORDED DURING THE MAIN STUDY III

 

Challenge Exposure: Control Group

ANIMAL NUMBER Sex: male

SKIN REACTION

24 hours

48 hours

After the patch removal

Left side

Right side

Left side

Right side

1

0

0

0

0

2

0

0

0

0

3

0

0

0

0

4

0

0

0

0

5

0

0

0

0

Challenge Exposure: Test Group

ANIMAL NUMBER Sex: male

SKIN REACTION

24 hours

48 hours

After the patch removal

Left side

Right side

Left side

Right side

3

0

0

0

0

6

0

0

0

0

7

0

0

0

0

8

0

0

0

0

9

0

0

0

0

10

0

0

0

0

11

0

0

0

0

12

0

0

0

0

14

0

0

0

0

15

0

0

0

0

Scoring of Skin Sensitisation

0 = no visible change

1 = discrete or patchy erythema

2 = moderate and confluent erythema

3 = intense erythema and swelling

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, under the conditions of the present assay the test item Reactive Red F03-0318 (Batch No.: HAS 366302) has shown to have no sensitisation potential. Consequently, Reactive Red F03-0318 has not to be classified according to current EU-regulations.
Executive summary:

A skin sensitisation study was performed in the guinea pig according to the Magnusson-Kligman method, using a maximisation method with Freund's complete adjuvant to evaluate the sensitisation potential of test item Reactive Red F03-0318.

 

Study conducted to OECD Guidelines for Testing of Chemicals 406 (17 July 1992) OECD: Paris. Commission Regulation (EC) No 440/2008 of 30 May 2008; B.6 and US EPA OPPTS 870.2600 (EPA 712-C-03-197, March 2003). Study performed in compliance with the principles of Good Laboratory Practice (GLP) and reported with a valid GLP certificate.

 

Ten test animals were subjected to sensitisation procedures in a two-stage process, i.e. an intra-dermal treatment and a topical application. The test item was used at a concentration of 5 (w/v) % in physiological saline solution for intra-dermal injections (Main Study I) and at a concentration of 50 % (w/v) in distilled water for dermal sensitisation treatment (Main Study II). Two weeks after the last induction exposure, a challenge dose (at a concentration of 50 % (w/v) in distilled water) was administered on the guinea pigs' left flank (Main Study III). The right flank area of animals was treated with 50 % dilution with distilled water of the maximum dermal challenge dose as a safeguard dose (25 % (w/v) in distilled water). Challenge was performed by dermal application of the test item to the shaved skin.

 

Five control guinea pigs were simultaneously exposed to physiological saline (NaCl 0.9 %) during the intra-dermal induction phase (Main Study I). During the dermal induction phase (Main Study II), the control animals were treated with distilled water only. During the challenge (Main Study III), control animals were treated with the test item at a concentration of 50 % (w/v) and 25 % (w/v) in distilled water only during the challenge at the left and right shaved flank, respectively.

 

Incidence Rate:

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

 

Intensity of Sensitisation Response:

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

In conclusion, under the conditions of the present assay the test item Reactive Red F03 -0318 (Batch No.: HAS 366302) has shown to have no sensitisation potenial. Consequently, Reactive Red F03 -0318 has not to be classified according to current EU-regulations.