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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:
27 September 2011 to 6 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to OECD, EU and US test guidance in compliance with GLP and reported with a valid GLP certificate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Details on test material:
Name: Reactive Orange F08-0314

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: LAL/HA/BR
Sex:
male
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: Test groups: 10 animals, Control group: 5 animals
Sex: Male
Body weight range at the beginning of the study: 326 – 399 g
Age of animals at arrival: Young adult, 8 weeks
Acclimatization time: 15 days

Husbandry
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: 628/3
Light: 12 hours daily from 6 a.m. to 6 p.m. (artificial light)
Temperature during the main study: 20 ± 3°C
Relative humidity during the main study: 30 – 70 %
Ventilation: 15-20 air exchange/hour

The environmental parameters were recorded twice daily during the study.

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.

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.

Body Weight

The body weights of individual animals were recorded at the beginning (on the day of randomisation) and at the end of the experiment. The mean values and the standard deviations were calculated in the control and test groups.

Observations

Mortality: Daily from delivery of the animals to the termination of the test.
Clinical signs: Daily during the test.

Skin reactions were observed and recorded as described below:
- Preliminary study: 1, 24, 48 and 72 hours after the patch removal.
- Intra-dermal induction exposure: 24 hours after the treatment.
- Dermal induction exposure: 1, 24, 48 and 72 hours after the patch removal.
- Challenge exposure: 24 and 48 hours after the patch removal.



Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Remarks:
and distilled water
Concentration / amount:
Preliminary Dose Range Finding Study:
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 hair free skin. 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 hair free skin.

Main Study I: Intra-dermal Induction Exposure:
Test Groups:
2 injections with 0.10 ml of Freund's Complete Adjuvant mixed with physiological saline (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.

Main study II: 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.

Main study III: Challenge Exposure:
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).


Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Remarks:
and distilled water
Concentration / amount:
Preliminary Dose Range Finding Study:
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 hair free skin. 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 hair free skin.

Main Study I: Intra-dermal Induction Exposure:
Test Groups:
2 injections with 0.10 ml of Freund's Complete Adjuvant mixed with physiological saline (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.

Main study II: 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.

Main study III: Challenge Exposure:
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).


No. of animals per dose:
Test groups: 10 male animals, Control group: 5 male animals

Details on study design:
TEST PROCEDURE

Preliminary Dose Range Finding Study

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 hair free skin. 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 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 5, 1, 0.1 and 0.01 % (w/v) in Physiological saline solution resulted no reaction (scores 0-0) in the skin of guinea pigs.

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 a gauze swab saturated with a mild shampoo (Gabi bath – produced by Sara Lee –NL Fruitweg 25., 2525 KG The Hague) and water at body temperature (it was found that the water was adequate to clean the application site, only water was used in the main study).

It was found that 0.5 ml of the test formulation in distilled water at concentrations of 75 and 50 (w/v) % produced very slight erythema (score 1) at 1 hour after the patch removal. Concentrations of 25 and 10 % (w/v) resulted no reaction (scores 0) 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 intra-dermal treatment, the 5 (w/v) % in physiological saline solution was used.

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.

Main Study

STUDY DESIGN

The treatments, concentrations of the test item and the times of observations are detailed in Table 1 under any other information on materials and methods.

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 (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 50 % formulation of physiological saline in a 1:1 mixture (v/v) 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.

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. 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.

OBSERVATION AND SCORING

The dermal irritation scores (in case of preliminary study (primary irritation) and in case of dermal exposure) were evaluated according to the scoring system by Draize (1959) presented by the following table.

Erythema and eschar formation:
No Erythema: 0
Very slight Erythema (barely perceptible): 1
Well defined Erythema: 2
Moderate to severe Erythema: 3
Severe Erythema (beet redness) to slight eschar formation (injuries in depth): 4

Oedema formation:
No oedema: 0
Very slight oedema (barely perceptible): 1
Slight oedema (edges of area well defined by definite raising): 2
Moderate oedema (raised appr. 1 mm): 3
Severe oedema (raised more than 1 mm and extending beyond area of exposure): 4

CLASSIFICATION OF SKIN IRRITATION
0 = non irritant
1 = slightly irritant
2-3 = mildly to moderately irritant
4 = severely irritant

After the challenge exposure, each animal was examined and scored 24 and 48 hours after the end of the exposure period.

Grading was performed according to the following system:

CLASSIFICATION OF SKIN SENSITISATION
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

CLASSIFICATION OF SENSITIZER

The results obtained for test animals at the challenge application were compared with those simultaneously obtained in control animals.

A test animal was considered to show evidence of contact hypersensitivity if the observed dermal reaction at challenge is more marked and/or persistent than dermal reaction seen in animals of the control group. If dermal scores of 1 were noted in control animals and the dermal reactions of the test group animals were not clearly different from the reaction in the animals of the control group, the test results were classified as “inconclusive”.

A test animal was considered to show no evidence of contact hypersensitivity if the dermal reaction resulting from the challenge application is identical or less marked and/or persistent than any dermal reaction seen in animals of the control group.

The percentage of animals showing a positive reaction was calculated in both (test and control) groups. As a result of these, the percentage value of the control animals that responded was subtracted from the percentage of the test animals responding positively to the challenge. The net response value is the percent sensitisation. According to the Commission Regulation (EC) No 440/2008 of 30 May 2008; B.6, if at least 30 % of the animals show allergic response, after the Magnusson and Kligman procedure, the test item will be classified as a "sensitizer".

RELIABILITY STUDY

The sensitivity and reliability of the experimental procedure is assessed twice a year by use of items which are known to have moderate skin sensitisation properties such as 2-Mercaptobenzothiazole (OECD 406, adopted 17 July 1992 chapter 10., 11.).

The results of the latest reliability check (Study Code: 11/112-104T) were as follows:

START OF EXPERIMENT : 09 May 2011
END OF EXPERIMENT : 10 June 2011
DATE OF FINAL REPORT : 24 June 2011

The selection of dose levels was made on the basis of the previous reliability study.

In the reliability study, the test animals were treated with the reference item as follows:

Intra-dermal induction exposure: 1% (w/v)
Dermal treatment: 75% (w/v)
Challenge treatment: 50% (w/v)

REFERENCE ITEM AND VEHICLE

Test Item: 2-Mercaptobenzothiazole
Lot number: MKBF3725
CAS Number: 149-30-4
Expiry date: 11 May 2012
Manufacturer: Sigma-Aldrich Co.
Storage condition: Room temperature

Component of vehicle:

Name: Methylcellulosum
Synonym: Methylcellulose
Batch number: K93935287 / O16147824
Manufacturer: DOW CHEMICALS
Expiry Date: May 2012 / 21 September 2014
Storage condition: Room temperature

Name: Distilled water
Batch No.: 0110111
Manufacturer: TEVA Co.
Expiry: January 2014
Storage condition: Room temperature

Subsidiary material:

Name: Freund's complete adjuvant (FCA)
LOT number: 080M8723
Expiry date: 08 February 2012
Produced by: Sigma-Aldrich Co.

Name: Physiological saline solution (NaCl 0.9 %)
Batch/lot number: 3390210
Date of expiration: February 2013
Produced by: TEVA Co.


SUMMARY OF THE RELIABILITY STUDY

Challenge with test item 2-Mercaptobenzothiazole resulted in a positive response in test animals sensitised previously. The net response values at the 24 and 48 hours observations represented an incidence rate of 50 % and 40 % and the net score values of 0.70 and 0.40 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 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.




Challenge controls:
As above.
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole

Study design: in vivo (LLNA)

Concentration:
Not applicable.
No. of animals per dose:
Not applicable.
Details on study design:
Not applicable.
Statistics:
Not applicable.

Results and discussion

Positive control results:

Challenge with test item 2-Mercaptobenzothiazole resulted in a positive response in test animals sensitised previously. The net response values at the 24 and 48 hours observations represented an incidence rate of 50 % and 40 % and the net score values of 0.70 and 0.40 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 erythema developed on the skin of sensitised guinea pigs.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
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 chemical
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:
negative 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: negative control. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative 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: negative control. Dose level: 75%. No with. + reactions: 0.0. Total no. in groups: 5.0.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Not applicable
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable

Any other information on results incl. tables

Main Study

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 and at a concentration of 75 % (w/v) in distilled water for dermal sensitisation treatment.Two weeks after the last induction exposure, a challenge dose (at a concentration of 75 % (w/v) in distilled water) was administered on the left flank of animal. 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.

Five control guinea pigs were simultaneously exposed to Physiological saline (NaCl 0.9 %) during the sensitisation phase I (intra-dermal treatment). During the sensitisation phase II (dermal treatment) the control animals were treated with distilled water and they were treated with the test item at a concentration of 75 % (w/v) and 37.5 % (w/v) in distilled water only during the challenge.


Skin Effects after the Challenge Exposure

 

Test group

 

After the challenge with the test itemat a concentration of 75 % (w/v) in distilled water, no positive response was observed in the treated animals. The mean of the scores was 0.00 according to the 24 and 48-hours results. The right shaved flank area of all animals was treated with a test item concentration of 37.5 (w/v) % in distilled water as a safeguard and no reaction was noted.

 

Control group

 

After the challenge with the test itemat 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 was treated with a test item concentration of 37.5 (w/v) % in distilled water as a safeguard and no reaction was noted.

Clinical Observations

There were no overt signs of an adverse clinical response to treatment with the test item during the course of the study.

Mortality

There were no moribund or dead animals during the study.

 

Body Weight

The individual body weights of the guinea pigs were measured at the day of randomisation and at the end of experiment. There were no notable differences between the test animal group and the control group.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Challenge with test item Reactive Orange F08-0314 evoked no positive responses in the test animals sensitised previously with the test item or in the control group. The net response value represented an incidence rate of 0 % and the net score value of 0.00.

In conclusion, under the conditions of the present assay the test item Reactive Orange F08-0314 (Batch No.: HAS 365803) was shown to have no sensitisation potential and classified as a non-sensitizer, 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 Orange F08-0314.

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 and at a concentration of 75 % (w/v) in distilled water for dermal sensitisation treatment. Two weeks after the last induction exposure, a challenge dose (at a concentration of 75 % (w/v) in distilled water) was administeredon the left flank of animal.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.

Five control guinea pigs were simultaneously exposed to Physiological saline (NaCl 0.9 %) during the sensitisation phase I (intra-dermal treatment). During the sensitisation phase II (dermal treatment) the control animals were treated with distilled water and they were treated with the test itemat a concentration of 75 % (w/v )and 37.5 % (w/v) in distilled water only during the challenge.

 

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.