Disodium 4-[4-[[5-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]-2,5-dichlorobenzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Data from a reliable in vivo test conducted before the enforcement of Commission Regulation (EU) 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) are available.

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4-[4-[[5-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]-2,5-dichlorobenzenesulphonate

EC Number:

274-499-0

EC Name:

Disodium 4-[4-[[5-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]-2,5-dichlorobenzenesulphonate

Cas Number:

70247-70-0

Molecular formula:

C19H14BrCl2N5O8S2.2Na

IUPAC Name:

disodium 4-[4-({5-[(2-bromoacryloyl)amino]-2-sulfonatophenyl}diazenyl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl]-2,5-dichlorobenzenesulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DUW 1410/21
- Expiration date of the lot/batch: June 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature (approx. 20 °C),
away from direct sunlight
- Stability under test conditions: Stable for 24 hours
- Solubility and stability of the test substance in the solvent/vehicle: Stable in bi-distilled water, FCA and saline for 24 hours

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Test system: Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF
- Rational: Recognized by the international guidelines as a recommended test system (e.g. OECD, EEC).
- Source: Harlan Nederland B.V.
Postbus 167
NL-3700 AD Zeist / The Netherlands
Woundenbergseweg 55
NL-3707 HW Zeist / The Netherlands
- Age at delivery: 5-7 weeks
- Age at beginning of pre-test/acclimatization period: 6-8 weeks
- Body weight at pretest start: Pretest group: 406-448 gr
- Body weight at beginning of acclimatization period: Control and test group: 425-510 gr.
- Identification: By unique cage number and corresponding ear tags.
- Randomization: Randomly selected at time of delivery.
- Diet : Pelleted standard Nafag Ecosan 845 25W4, batch nos. 37/97 and 58/97 guinea pig breeding / maintenance diet ("Nafag",
Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum.
- Water: Community tap water from Itingen, ad libitum. Once weekly additional supply of ascorbic acid (approx. 1 g/1) via the drinking water was provided.
- Acclimation period: One week for the control and test group under test conditions after health examination. One week for the animals of the pretest. Only animals without any visible signs of illness were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15 (Air-conditioned)
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hours dark cycle. Music was played during the light period.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

5 %

Day(s)/duration:

Day 1

Adequacy of induction:

highest technically applicable concentration used

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

other: bi-distilled water

Concentration / amount:

50 %

Day(s)/duration:

Day 8

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

bi-distilled water

Concentration / amount:

25%

Day(s)/duration:

Day 22

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Number of animals for main study / pretest: 15 females / 3 females , nulliparous and non-pregnant.
- Control group: 5 animals (main study; animal number 535-539)
- Test group: 10 animals (main study; animal number 540-549)
- Intradermal pretest: 1 animal; animal number 550
- Epidermal pretest: 2 animals; animal number 551-552

Details on study design:

PRETEST/PERFORMED DURING ACCLIMATIZATION PERIOD:
The procedure employed for these investigations was as follows:

INTRADERMAL INJECTIONS:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of 5, 3 and 1 % of the test article in bi-distilled water.
The resulting dermal reactions were assessed 24 hours later. For intradermal induction application in the main study a 5 % test article concentration was selected

EPIDERMAL APPLICATIONS:
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (2x2 cm) were saturated with the test article at A = 50 % (this concentration was found to be the most qualified to assure an optimum technical apphcation procedure), B = 25 %, C = 15 % and D = 10 % in bi-distilled water and applied to the clipped and shaved flanks. The volume of test article applied at 50 % was approximately 0.2 g. The volume of the remaining test article concentrations was 0.2 ml. Thepatches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24hours.
Approximately 21 hours after removal of the dressing the apphcation site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) to clean the apphcation site from staining produced by the test article, so that possible erythema reactions were clearly visible at that time.

The depilatory cream was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize described above.
The position of the epidermal applications is shown below:

Cranial
-----------
A C
left right
B D
------------
Caudal


Cranial
-----------
D B
left right
A C
------------
Caudal
The allocation of the different test dilutions to the sites (A, B, C, D) on the animals was alternated in order to minimize site-to-site variation in responsiveness. The concentration selected for the induction period and challenge procedure was 50 % and 25 %, respectively.


MAIN STUDY
INTRADERMAL INJECTIONS / PERFORMED ON TEST DAY 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:

TEST GROUP:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, diluted to 5 % with bi-distilled water.
3) The test article diluted to 5 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

CONTRAOL GROUP:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Bi-distilled water.
3) 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

EPIDERMAL APPLICATIONS / PERFORMED ON TEST DAY 8
On test day 7 and approximately 20 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) of the animals of the control and test group was clipped, shaved free of hair and the test area was pretreated with a 10 % dilution of Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass rod without bandaging. This 10 % concentration of SLS enhances sensitization by provoking a mild inflammatory reaction (Magnusson and Kligman 1970).
One week after the injections, the scapular area (approximately 6 x 8 cm) was again cupped and shaved free of hair. A 2 x 4 cm patch of filter paper was saturated with the test article (50 % in bi-distilled water) and placed over the injection sites of the test animals. The volume of test article applied was approximately 0.3 g. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test article.
The guinea pigs of the control group were treated as described above with bi-distilled water only.
Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.


B. CHALLENGE EXPOSURE/PERFORMED ON TEST DAY 22

The test and control guinea pigs were challenged two weeks after the epidermal induction application. The test and control guinea pigs were treated in the same way. Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (2x2 cm) of filter paper were saturated with the highest non-irritating concentration of 25 % (left flank) and the vehicle only (bi-distilled water applied to the right flank) using the same method as for the epidermal application. The volume of test article applied was approximately 0.2 ml. The dressings were left in place for 24 hours. Approximately 21 hours after removal of the dressing the test sites treated with the test article were depilated as described in the epidermal pretest. Approximately 24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.

INTERPRETATION:
The results obtained from test animals following the challenge applications were compared with the results seen in control animals. An allergic reaction was defined by visible reddening of the challenge site. If the dermal reactions of test animals following the challenge were more marked and/or persistent than those of the control animals, the animals were considered to show evidence of contact hypersensitivity. If the dermal reactions of test animals following the challenge were not clearly different from the reactions seen in the control group animals, the results for the test animals were considered "inconclusive". The test animals were considered to show no evidence of contact hypersensitivity if the dermal reactions to the challenge application were identical to or less marked and/or persistent than the reactions observed in the control animals.
By "maximizing" the exposure and enhancing allergenicity, some problems could arise, particularly in relation to specificity, especially the potential for false-positive reactions. An inflammatory response at challenge may not necessarily be due to allergenicity, but instead may be a false-positive irritant response caused by an inducing hyperirritability.

Challenge controls:

Control guinea pigs were challenged two weeks after the epidermal induction application. Fur was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (2x2 cm) of filter paper were saturated with the highest non-irritating concentration of 25 % (left flank) and the vehicle only (bi-distilled water applied to the right flank) using the same method as for the epidermal application. The volume of test article applied was approximately 0.2 ml. The dressings were left in place for 24 hours.

Approximately 21 hours after removal of the dressing the test sites treated with the test article were depilated as described in the epidermal pretest.
Approximately 24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.

Positive control substance(s):

yes

Remarks:

Alpha-hexylcinnalmaldehyde

Results and discussion

Positive control results:

In this study 70 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 25 % in polyethylene glycol (PEG 400). No skin reactions were observed in the control group. A response of at least 30 % positive animals is considered positive "R43": may cause sensitization by skin contact according to the "Commission Directive 96/54/EEC, July 30, 1996 adapting to technical progress for the 22nd time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances". Therefore, the test article ALPHA-HEXYLCINNAMALDEHYDE applied at a concentration of 25 % in polyethylene glycol (PEG 400) is considered to be a sensitizer when used under the described test conditions.

According to the rating of allergenicity by Magnusson and Kligman the test article is a strong sensitizer.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

ALPHA-HEXYLCINNAM ALDEHYDE, intradermal induction: 5 %, epidermal induction: 50 % and challenge 25 %

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

ALPHA-HEXYLCINNAM ALDEHYDE, intradermal induction: 5 %, epidermal induction: 50 % and challenge 25 %

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Intradermal induction: 5 %, epidermal induction: 50 % and challenge 25 %

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Intradermal induction: 5 %, epidermal induction: 50 % and challenge 25 %

No. with + reactions:

8

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Vehicle

No. with + reactions:

0

Total no. in group:

5

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Vehicle

No. with + reactions:

0

Total no. in group:

5

Any other information on results incl. tables

SKIN EFFECTS AFTER EPIDERMAL INDUCTION - PERFORMED ON TEST DAY 8

CONTROL GROUP

Erythematous reaction was observed in the animals (nos. 535-538; at the 24-hour reading) and (nos. 536 and 538; at the 48-hour reading) when treated with bi-distilled water only.

TEST GROUP

Erythematous reaction was observed in the animals (nos. 541-547 and 549; at the 24-hour reading) and (nos. 541-542, 544-547 and 549; at the 48-hour reading) when treated with the test article at 50 % in bi-distilled water.

Even so the test article stained the skin slightly yellow, it was possible to determine whether erythema was present or not. However no edema was observed.

All animals of the control and test group were pretreated with 10 % SLS in paraffinum perliquidum. 

 

SKIN EFFECTS AFTER THE CHALLENGE - PERFORMED ON TEST DAY 22

CONTROL GROUP

No positive reactions were observed in the animals either when treated with bi-distilled water only or when treated with the test article at 25 % in bi-distilled water.

Yellow discoloration was noted directly after removal of the patch. To remove discoloration all animals were depilated approximately 3 hours prior to challenge reading.

TEST GROUP

Positive reactions were observed in seven out of 10 animals (nos. 540, 542-543, 545-547 and 549; at the 24-hour reading) and in eight out of 10 animals (nos. 540, 542-547 and 549; at the 48-hour reading) when treated with the test article at 25 % in bi-distilled water. No positive reactions were observed when treated with bi-distilled water only.

Yellow discoloration was noted directly after removal of the patch. To remove discoloration all animals were depilated approximately 3 hours prior to challenge reading.

 

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

As there were no deaths during the course of the treatment period no necropsies were performed.

 

CLINICAL SIGNS, SYSTEMIC

No symptoms of systemic toxicity were observed in the animals.

 

BODY WEIGHTS

The female animal no. 546 showed a slight loss of body weight during the treatment/observation period. The body weight of the other animals was within the range of physiological variability known for guinea pigs of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

FAT 40061/E is considered as a skin sensitiser in the contact hypersensitivity test in albino guinea pigs (Maximization test).

Executive summary:

A study was performed as per OECD 406 guideline to assess the skin sensitization potential of FAT 40061/E using the Guinea pig maximisation method. Based on the findings of the pre-test, 5, 50 and 25 % test concentrations were used for intradermal induction, epidermal induction and challenge, respectively. In this study, 70 and 80 % of the animals of the test group were observed with positive skin reactions challenge. No skin reactions were observed in the control group. Therefore, the test article FAT 40061/E is considered to be a sensitizer when used under the described test conditions.