Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxy-1-naphthalenesulfonato(3-)]-, disodium

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 06 to May 02, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

yes

Remarks:

In the Intradermal induction of the main study the vehicle is mixed with FCA only. A mixture of FCA:physiological saline was chosen instead of FCA only in order to decrease the site effects of FCA when applied alone.

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A LLNA study has not been conducted because adequate data from guinea pig Maximisation test study are already available.

Test material

Test material information

Constituent 1

Reference substance name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxy-1-naphthalenesulfonato(3-)]-, disodium

EC Number:

281-013-0

EC Name:

Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-3-hydroxy-1-naphthalenesulfonato(3-)]-, disodium

Cas Number:

83833-37-8

Molecular formula:

C36H24CrN9Na2O9S

IUPAC Name:

hydroxylamine

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Ibm: GOHI; SPF-quality guinea pigs

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, CH-4414 Füllinsdorf/Switzerland
- Age at beginning of Acclimatization period: 6 - 8 weeks
- Body Weight at beginning of Acclimatization period: Control and Test Group 315 - 422 g; Pretest 334 - 420 g
- Identification: by unique cage number and corresponding ear tags.
- Randomization: randomly selected at time of delivery
- Housing: individually in Makrolon type-3 cages (size: 22x37x15 cm) with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
- Diet: pelleted standard Kliba 342, Batch nos. 61/94 (at delivery of the animals to 07-APR-1994) and 62/94 (from 08-APR-1994 to termination of test) guinea pig breeding/ maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum
- Water: community tap water from Füllinsdorf, ad libitum. Once weekly additional supply
of ascorbic acid (1 g/1) via the drinking water.
- Acclimation period: one week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest.
- Indication of any skin lesions: Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 20-23 °C
- Humidity (%): between 36.5 - 78 %
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light (approx. 100 Lux) /12 hours dark
- Cleaning process: the duration of the cleaning process was approximately 30 minutes

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

other: For intradermal application: Freund's Complete Adjuvant, physiological saline and Bi-distilled water; for Epidermal application: Vaselinum album

Concentration / amount:

For intradermal application 0.1 ml of 1% of test article solution; for Epidermal application 25 % of test article in vaselinum album.

Day(s)/duration:

Intradermal Induction performed at Day 1; Epidermal induction performed at Day 8

Challenge

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

5 % of test item in vaselinum album

Day(s)/duration:

Test performed at Day 22

No. of animals per dose:

Number of animals for pretest: 6
Number of animals for main study: 30

Details on study design:

RANGE FINDING TESTS
INTRADERMAL INJECTIONS
Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs 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 a 1% test article dilution was selected.
EPIDERMAL APPLICATIONS
Both flanks of each of 4 guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches* of filter paper ( 2 x 2 cm) were saturated
with the test article at A = 25 % (this concentration used was found to be the most qualified to assure an optimum technical application procedure), B = 15 %,
C = 10 % and D = 5 % of the test article in vaselinum album and applied to the clipped and shaved flanks. The patches 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 24 hours. Approximately 20.5 hours after removing of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) to clean the application site from violet staining produced by the test article, so that possible erythema reactions were clearly visible at that time.
The depilatory 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. 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.
For the epidermal induction the test article at 25% and for the challenge procedure the test article at 5% was selected.

MAIN STUDY
A. INDUCTION EXPOSURE
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 1% with bi-distilled water.
3) The test article diluted to 1% by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Control Group: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Bi-disti1 led 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:
One week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair. A 2 x 4 cm patch of filter paper was
saturated with the test article (25 % in vaselinum album) and placed over the injection sites of the test animals. 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 approximately 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 vaselinum album 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
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 ( 2 x 2 cm) of filter
paper were saturated with the highest non-irritating concentration of 5 % (left flank) and the vehicle only (vaselinum album, applied to the right flank) using
the same method as for the epidermal application. The dressing were left in place for 24 hours.
21 hours after removing of the dressing the test sites were depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil). The cream was placed on the patch sites for 3-5 minutes and then washed off with a stream of warm running water. When the application sites were clean and any stains from the test article removed the animals were dried with a disposable paper towel and returned to their cages.
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:

4-AMINOBENZOIC ACID ETHYL ESTER 25 % IN MINERAL OIL

Results and discussion

Positive control results:

Moderate allergenic potency (grade 3) of the test article 4-AMINOBENZOIC ACID ETHYL ESTER.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5 % in vaselinum album

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No toxic symptoms

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5 % in vaselinum album

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No toxic symptoms

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

only vehicle

No. with + reactions:

0

Total no. in group:

10

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

only vehicle

No. with + reactions:

0

Total no. in group:

10

Results 5

Group:

positive control

Remarks on result:

other: not reported

Any other information on results incl. tables

CONTROL GROUP

Injection site 1 (1:1 (v/v) mixture of Freund's Complete Adjuvant [FCA] and physiological saline)

The area around the injection site was oedematous and erythematous from test day 2 to 5 followed by necrosis, encrustation and exfoliation of the encrustation up to the termination of test (test day 27).

Injection site 2 (bi-distilled water)

The area around the injection site was oedematous and erythematous from test day 2 to 4.

Injection site 3 (1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of FCA and physiological saline)

The reactions observed were identical to those obtained at injection site 1 with the mixture of FCA and physiological saline.

As the animals were bandaged with the semi-occlusive dressing no observations of the skin were possible on test day 9.

TEST GROUP

Injection site 1 (1:1 (v/v) mixture of Freund's Complete Adjuvant [FCA] and physiological saline)

The reactions observed were identical to those obtained in the control group with the mixture of FCA and physiological saline, at injection site 1.

Injection site 2 (1% solution of test article in bi-distilled water)

The area around the injection site was oedematous and violet discoloured from test day 2 to 4 and 2 to 8 respectively and became necrotic from test day 5 to 12 followed by encrustation and exfoliation of encrustation up to test day 27.

Injection site 3 (1% solution of test article in a 1:1 (v/v) mixture of of FCA and physiological saline)

The reactions observed were identical to those obtained at the injection site 2 with a 1% solution of test article in bi-distilled water. As the animals were bandaged with the semi-occlusive dressing no observations of the skin were possible on test day 9.

SKIN EFFECTS AFTER EPIDERMAL INDUCTION PERFORMED ON TEST DAY 8

CONTROL GROUP: No erythematous or oedematous reaction was observed in the animals treated with vaselinum album only. TEST GROUP: As the test article stained the skin violet, it was not possible to determine whether erythema was present. However, no oedema was observed. Violet discolouration was noted from test day 10 to 23.

SKIN EFFECTS AFTER THE CHALLENGE PERFORMED ON TEST DAY 22

CONTROL and TEST GROUP: No positive reactions were observed in the animals either when treated with vaselinum album alone or when treated with the test article at 5% in vaselinum album. Violet discolouration was noted from test day 23 (after removal of the dressing) to 25.

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

Two animals of the control group, 12 of the test, 2 of the intradermal pretest and all animals of the epidermal pretest lost weight during the acclimatization period. No loss of weight was noted during the treatment period.

Applicant's summary and conclusion

Interpretation of results:

other: CLP criteria not met

Conclusions:

Non sensitising

Executive summary:

Method

To assess the allergenic potential of the test substance in albino guinea pigs the Maximization-Test of B. Magnusson and A.M. Kligman (1969) was used, according to the OECD guideline 406. Ten males were used as control group and 20 males were used as test group.

The highest non-irritating test article concentration used for challenge application was 5% in vaselinum album.

Results

0% of the animals were positive at the 24- and 48-hour reading respectively after treatment with the highest non-irritating test substance concentration of 5% in vaselinum album. No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred.

Conclusion

Non sensitising.