Chromate(5-), bis[4-(hydroxy-κO)-7-[2-(2-hydroxy-1-naphthalenyl)diazenyl]-3-[2-[2-(hydroxy-κO)- 3-nitro-5-sulfophenyl]diazenyl]-2-naphthalenesulfonato(4-)]-, sodium (1:5)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study (different from LLNA test) is available.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

415-070-1

EC Name:

-

Molecular formula:

C52H26CrN10Na5O22S4

IUPAC Name:

chromium(3+) pentasodium bis(6-[2-(2-hydroxynaphthalen-1-yl)diazen-1-yl]-2-[2-(3-nitro-2-oxido-5-sulfonatophenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate)

In vivo test system

Test animals

Species:

guinea pig

Strain:

Himalayan

Remarks:

SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
-Source: BRL Ltd., Basel, Switzerland
-Age at study initiation: ~ 9 weeks
-Weight at study initiation: 353 - 490 g
-Housing: Group housing of 2 animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands).
-Diet: Free access to standard guinea pig diet, including ascorbic acid (1600 mg/kg);
LC 23-B, pellet diameter 4tnm (Hope farms, Woerden, The Netherlands).
-Water: Free access to tap water, diluted with decalcified water.
-Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
-Temperature (°C): 21 °C
-Humidity (%): 55 %
-Air changes (per hr): 15 air changes per hour
-Photoperiod (hrs dark / hrs light): 12 hour cycle dark/light with a fluorescent light

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

water

Concentration / amount:

0.5 % (w/w)

Day(s)/duration:

a single injection

Induction 2

Route:

intradermal

Vehicle:

other: 50:50 FCA distilled water

Concentration / amount:

0 %

Day(s)/duration:

a single injection

Induction 3

Route:

intradermal

Vehicle:

other: 50:50 FCA distilled water

Concentration / amount:

1 %

Day(s)/duration:

a single injection

Induction 4

Route:

epicutaneous, semiocclusive

Vehicle:

water

Concentration / amount:

25 %

Day(s)/duration:

48 hours

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

0.05 ml of 50%, 25% and 10%

Day(s)/duration:

24 hours

No. of animals per dose:

Preliminary study : 5 females
Experimental group : 20 females
Control group : 10 females

Details on study design:

PRELIMINARY STUDY
INDUCTION EXPOSURE: INTRADERMAL INJECTIONS
-No. of exposures: four injections
-Test groups: one female guinea pig
-Control group: none
-Site: clipped shoulder regions.
-Concentrations: 0.5 % (w/w) in distilled water.
-Evaluation (hr after challenge): 24 and 48 hours
INDUCTION EXPOSURE: EPIDERMAL APPLICATION
-No. of exposures: one application
-Exposure period: 24 hours
-Test groups: one female guinea pig (the same animal of intradermal injection)
-Control group: none
-Site: shaved flank
-Protection site: Scotchpak-non-woven patch on Micropore tape (both 3M, St. Paul, USA) and held in place by Coban elastic bandage (3M, St. Paul, USA).
-Concentrations: 50% (w/w) test substance concentration in distilled water (0.5 ml) was applied epidermally
-Evaluation (hr after challenge): 24 and 48 hours
EPIDERMAL APPLICATIONS
-No. of exposures: one
-Exposure period: 24 hours
-Test groups: four female guinea pig
-Control group: none
-Site: shaved flank
-Protection site: Square chambers (v.d. Bend, Brielle, The Netherlands), mounted on Micropore tape (3M, St. Paul, USA) and held in place by Coban elastic bandage.
-Concentrations: Four concentrations of the test substance in distilled water (50 %, 25 %, 10 % and 5 % w/w, 0.05 ml each)
-Evaluation (hr after challenge): 24 and 48 hours

The erythema and oedema assessments were based on this scale:
-Erythema and eschar formation:
No erythema: 0
Slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate erythema: 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth): 4
-Oedema:
No oedema: 0
Slight oedema (barely perceptible): 1
Well-defined oedema (edges of area well-defined by definite raising): 2
Moderate oedema (raised approximately 1 mm): 3
Severe oedema (raised more than 1 mm and extending beyond the area of exposure): 4

MAIN STUDY
A1. INDUCTION EXPOSURE: INTRADERMAL INJECTIONS
-No. of exposures: hree pairs of injections (0.1 ml/site)
-Exposure period: single injection
-Test groups: 20 female guinea pigs
-Control group: 10 female gunea pigs
-Site: scapular region
-Frequency of applications: one application for each site
-Concentrations:
A) The test substance at a 0.5% (w/w) concentration in physiological saline.
B) Freunds' complete adjuvant 50:50 with water for injection (Ferensius AG, Bad Homburg, Germany).
C) The test substance, at twice the concentration used in (A), emulsified in a 50:50 mixture of Freunds' complete adjuvant (Difco,
Brunschwig, Amsterdam, The Netherlands).

A2. INDUCTION EXPOSURE: EPIDERMAL APPLICATION (after 7 days)
The clipped scapular area was rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, The Netherlands) in petrolatum using a spatula.
This concentration of SDS enhances sensitisation by provoking a mild inflammatory reaction.
-No. of exposures: one application
-Test groups: 20 female guinea pigs
-Control group: 10 female gunea pigs
-Site: scapular area
-Protection site: Scotchpak-non-woven patch mounted on Micropore tape and secured with Coban elastic bandage.
-Frequency of applications: one application
-Duration: 48 hours
-Concentrations: 0.5 ml of a 25% (w/w) test substance concentration

B. CHALLENGE EXPOSURE (after 14 days)
-No. of exposures: three applications
-Exposure period: 24 hours (excepted for animal 288 that had removed dressing before the 24 hour application period)
-Test groups: 20 female guinea pigs
-Control group: 10 female gunea pigs
-Site: flank
-Protection site: Square chambers attached to Micropore tape and secured with Coban elastic bandage.
-Concentrations: 0.05 ml of 25%, 10% concentration and the vehicle and approx. 0.05 ml for 50%
-Evaluation (hr after challenge): 24 and 48 hours after removal of the dressings.

The sites after removal were assessed for redness and swelling 24 and 48 h after removal of the dressings, using this numerical grading system:
no skin reaction: 0
Red spots (scattered reactions): 1
Moderate but confluent redness: 2
Redness and swelling: 3
Intense reddening and swelling: 4
The test sites were shaved with an electric razor after the first reading. All animals were killed at the
end of the test period by carbon dioxide asphyxiation.

Challenge controls:

yes

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

No. with + reactions:

2

Total no. in group:

20

Remarks on result:

other: This site was blue discoloured, which made skin reading difficult

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

4

Total no. in group:

20

Remarks on result:

other: in some case the site was blue discoloured, which made skin reading difficult.

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10 %

No. with + reactions:

2

Total no. in group:

20

Remarks on result:

other: in some case the site was blue discoloured, which made skin reading difficult.

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 %

No. with + reactions:

3

Total no. in group:

20

Results 5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

11

Total no. in group:

20

Results 6

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10 %

No. with + reactions:

8

Total no. in group:

20

Any other information on results incl. tables

Preliminary Study

No signs of systemic toxicity were observed during the preliminary study.

However, body weight loss was noted in one of the five animals. Selection of the test substance concentrations used in the main study was based on the findings in the preliminary study and in accordance with Magnusson and Kligtnan (1969).

Main Study

Induction: the experimental animals showed no visual erythema or oedema after the 48 hours occluded epidermal induction exposure. The treated skin area of all animals was blue discoloured by the test substance.

Main Study Challenge

-Control Group: No skin reactions were evident after the challenge exposure, with the exception of red spots noted in response to the 10% test substance concentration in one animal. A few control animals showed blue discolouration of the treated skin sites exposed to the 25 % and 10 % concentrations.

-Experimental Group: Three, twelve and eight animals showed red spots in response to the 50 %, 25 % and 10 % test substance concentrations, respectively. The majority of experimental animals showed blue discolouration of the treated skin sites exposed to the 25 % and 10 % concentrations and a few animals al so on the treated skin sites exposed to the 50 % concentration.

Toxicity Symptoms / Mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study during the study period.

Body Weight

The average body weight gain of experimental and control animals was similar.

Applicant's summary and conclusion

Interpretation of results:

other: Classified according to the CLP Regulation (EC 1272/2008) Skin sens. Cat. 1A

Conclusions:

The substance has sensitising properties

Executive summary:

The skin sensitisation potential was evaluated with an in vivo maximization test on guinea pigs, according to the method B.6 of EEC-Directive 92/69 EEC. A preliminary study was performed in order to determine the test concentrations of the main study. The concentrations 0, 10, 25 and 50 % were tested on 20 animals by intradermal (first induction) and the epidermal routes of administration (second induction and challenge). The readings after the challenge were at 24 and 48 hours. Three, twelve and eight animals showed positive reactions (redness score 1) to the 50%, 25% and 10% test substance concentrations, respectively. The majority of experimental animals showed blue discolouration of the treated skin sites exposed to the 25% and 10% concentrations and a few animals also on the treated skin sites exposed to the 50% concentration at 24-hour observation.