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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2011 to 17 December 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
The relative humidity (min. 25%) and temperature (max. 24.7oC) were outside of the target range (RH: 30 – 70%; T: 20 ± 3°C) during the study.
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
The relative humidity (min. 25%) and temperature (max. 24.7oC) were outside of the target range (RH: 30 – 70%; T: 20 ± 3°C) during the study.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
yes
Remarks:
The relative humidity (min. 25%) and temperature (max. 24.7oC) were outside of the target range (RH: 30 – 70%; T: 20 ± 3°C) during the study.
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study is not considered valid due to the reaction of the dye with the organic solvent. In a separate evaluation, a precipitate was observed when the test substance was mixed with the vehicle (N,N-dimethylformamide) used in this study.
Species:
guinea pig
Strain:
other: LAL/HA/BR
Sex:
male
Details on test animals and environmental conditions:
EXPERIMENTAL ANIMALS

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: females, 4 males
Main test: Test groups: 10 animals; Control group: 5 animals
Sex: Male
Body weight range at the beginning of the study: 257 – 342 g
Age of animals at arrival: Young adult, 8 weeks
Acclimatization time: 14 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: 602/4
Light: 12 hours daily from 6 a.m. to 6 p.m. (artificial light)
Temperature during the study: 20.0 – 24.7°C
Relative humidity during the 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: 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.

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.



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:
water
Concentration / amount:
approximately 0.5 ml of the 75 % (w/v) concentration of the test item in distilled water
Day(s)/duration:
Day 8 / 48 h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
left flank: 75% / 0.5 mL
reight flank: 37.5% /0.5 mL
Day(s)/duration:
Day 22 / 24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Preliminary test: 4 females, 4 males
Main test: Test groups: 10 male animals; Control group: 5 male animals
Details on study design:
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 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 % (w/v) produced very slight erythema (score 1) at 1 hour after the patch removal. Concentrations of 50, 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 were 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 as detailed below in table 1.

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
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.
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:
no indication of skin sensitisation
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:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
75%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
75%
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50%
No. with + reactions:
5
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:
50%
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

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 item at 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 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 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.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Challenge with test item Reactive Yellow F01-0555 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 Yellow F01-0555 (Batch No.: F01-0555-42 (DYWJ5976)) 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 Yellow F01-0555.

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.

 

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 Yellow F01-0555 (Batch No.: F01-0555-42 (DYWJ5976)) was shown to have no sensitisation potential and classified as a non-sensitizer, according to current EU-regulations.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Reactive Yellow F01-0555, tested in a suitable vehicle, was shown to have no sensitisation potential (sensitiser) in guinea pig using the Magnusson & Kligman Method.

In a Local Lymph Node Assay in the Mouse, stimulation index values for Reactive Yellow F01-0555 were 16.0, 7.0 and 1.0 at treatment concentrations of 25, 10 and 5 (w/v) %, respectively. Therefore, under the conditions of the assay, Reactive Yellow F01 -0555 was shown to have a sensitisation potential (sensitiser), however, this study is not considered valid due to the reaction of the dye with the organic solvent. In a separate evaluation, a precipitate was observed when the test substance was mixed with the vehicle (N,N-dimethylformamide) used in this study.


Migrated from Short description of key information:
Reactive Yellow F01-0555, tested in a suitable vehicle, was shown to have no sensitisation potential (sensitiser) in guinea pig using the Magnusson & Kligman Method.

Justification for selection of skin sensitisation endpoint:
Study conducted according to the Magnusson & Kligman Method is selected because the results are considered to be valid. A LLNA study was also conducted, however, the results were not considered valid as a result of the reaction of the dye with the organic solvent.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

The registered chemical is a reactive dye. For this class of dyes it was generally agreed between the members of the Ecological and Toxicological Association of Dyes and Organic Pigments Manufacturers (ETAD) that a possible risk for respiratory sensitisation for workers exists at high exposure.However the following should be noted:

 

1) For the substance no history of respiratory problems, such as occupational asthma, is associated with the manufacture and use of the specific substance.

 

2)Due to the granular form of the substance (spray dried in closed system from aqueous solution directly after synthesis) no risk for inhalative exposure arises.

 

The potential to cause respiratory sensitisation is therefore not considered to be applicable for this substance.

No evidence of respiratory sensitisation was noted in any of the studies conducted, and it is proposed that the substance is not a respiratory sensitiser.


Migrated from Short description of key information:
Not assessed. No evidence of respiratory sensitisation was noted in any of the studies conducted, and it is proposed that the substance is not a respiratory sensitiser.
 

Justification for selection of respiratory sensitisation endpoint:
The potential to cause respiratory sensitisation is not considered to be applicable for this substance. See explanation under discussion below.

Justification for classification or non-classification

The above study has been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the study was conducted to GLP and in compliance with agreed protocols. sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008).