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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 OCED, EC & 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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
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 F08-0146

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-ÁLL 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: 5 females, 4 males
Main test: Test groups: 10 animals; Control group: 5 animals
Sex: Male
Body weight range at the beginning of the study: 221 – 323 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 & Söhne GmbH+CO.KG, D-73494 Rosenberg, Germany) was available to animals during the study.
Animal room: 602/9
Light: 12 hours daily from 6 a.m. to 6 p.m. (artificial light)
Temperature during the main study: 20.0 – 23.9°C
Relative humidity during the main study: 25% – 58%
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: 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:
water
Concentration / amount:
Detailed in table form. Table attached under: Any other information on materials and methods incl. tables.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Detailed in table form. Table attached under: Any other information on materials and methods incl. tables.
No. of animals per dose:
Number of animals:
Preliminary test: 5 females, 4 males
Main test: Test groups: 10 animals; Control group: 5 animals
Sex: Male
Details on study design:
Justification of the dosages:
For the intra-dermal application, 0.1 mL of the formulated test item was injected at concentrations of 5, 1, 0.1 and 0.01 % (w/v) in Physiological saline solution (NaCl 0.9%), into the shaved skin of the flanks One concentration was injected on the right side and another concentration on the left side of the animal. Each concentration was injected in duplicate at different sites, so each animal received a total of four injections. Two or three animals were employed per concentration tested.

Following the intra-dermal administration, the exposed areas were covered for 24 hours with porous gauze fastened to protect the treatment area from physical damage caused by scratching etc.

It was found that 0.1 mL of the test item formulation administered at concentrations of 1, 0.1 and 0.01 % (w/v) in Physiological saline solution resulted no reaction (scores 0 and 0 for erythema and oedema, respectively) in the skin of guinea pigs. At concentration of 5 (w/v) % the test item coloured the injection site, no scab or wound was seen at the treatment area.

For the dermal application, approximately 0.5 mL of the formulated test item in distilled water was applied at concentrations of 75, 50, 25 and 10% (w/v) onto the clipped and shaved skin of the animals. The time of exposure for the dermal application was 24 hours. One concentration was used on the right side and another concentration on the left side of the animals. Two animals were used per concentration. The treated area was tinted by the test item. The remaining test item and the discoloration was washed with using a gauze swab and water at body temperature (it was found that the water was adequate to clean the application site).

It was found that 0.5 ml of the test formulation in distilled water at concentrations of 75, 50, 25 and 10 % (w/v) resulted no reaction (scores 0 and 0 for erythema and oedema, respectively) on the skin of guinea pigs.

On the basis of results of the Preliminary Dose Range Finding Study, the 75 % (w/v) in distilled water was used for dermal induction treatment. Control animals were treated with the vehicle (distilled water) only.

For the challenge exposure, all animals of the treatment and control group were treated with the 75 % (w/v) concentration and at concentration of 37.5 % (w/v) in distilled water as a safeguard dose.

For intra-dermal treatment, the 5 (w/v) % in physiological saline solution was used.
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.
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 cm2 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 Freund's Complete Adjuvant mixed with physiological saline (NaCl 0.9 %) (1:1) (v/v),
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 75 % (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 cm² on the left and right flank of each animal. A 5x5 cm² patch of sterile gauze was saturated with the test item at 75 % (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. 37.5 (w/v) % in distilled water).

The volume of formulated test item and vehicle was approximately 0.5 ml. The time of the exposure was 24 hours. After the patch removal any remaining test item was removed with a gauze swab.
Challenge controls:
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.
Positive control substance(s):
yes
Remarks:
Freund's Complete Adjuvant

Results and discussion

Positive control results:
After the challenge with the test item at a concentration of 75 % (w/v) in distilled water no visible changes were found at the 24 and 48 hours examinations. The right shaved flank area of control animals treated with a test item concentration of 37.5 (w/v) % in distilled water as a safeguard showed no adverse effects.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75%
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: 75%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75%
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: 75%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
75%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
75%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 5.0.

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 F08-0146 (Batch No.: F08-0146-32) has shown to have no sensitisation potential. Consequently, Reactive Red F08-0146 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 F08-0146.

 

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 in compliance with the Principles of Good Laboratory Practice (GLP) and reported with a valid 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 75 % (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 75 % (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 (37.5 % (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), thecontrol animals were treated with distilled water only. During the challenge (Main Study III),control animalswere treated with the test item at a concentration of 75 % (w/v) and37.5 % (w/v) in distilled water 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 F08-0146 (Batch No.: F08-0146-32) has shown to have no sensitisation potential. Consequently, Reactive Red F08-0146 has not to be classified according to current EU-regulations.