Reaction mass of sodium(E)-6,7'-carbonylbis(azanediyl)bis(4-hydroxy-3-((E)-phenyldiazenyl) naphthalene-2-sulfonate) and disodium 3-[(4-acetamidophenyl)azo]-4-hydroxy-7-[[[[5-hydroxy-6-(phenylazo)-7-sulphonato-2-naphthyl]amino]carbonyl]amino]naphthalene-2-sulphonate and disodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-(phenylazo)naphthalene-2-sulphonate]

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

other: experimental data on similar substance

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Valid guideline used

Species:

guinea pig

Strain:

Himalayan

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wolferstrasse 4, CH-4414 Fullinsdorf
- Age at study initiation: males: 7 weeks, females: 8 weeks
- Weight at study initiation: males: 308 - 355 g, females: 297 - 412 g
- Housing: Individually in Makrolon type-3 cages
- Diet: Pelleted standard Kliba 342, Batch 57/90 guinea pig breeding/maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum
- Water: tap water ad libitum; once weekly additional supply of ascorbic acid via the drinking water
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal and epicutaneous

Vehicle:

physiological saline

Concentration / amount:

Intradermal induction: 3%
Epicutaneous induction: 15%
Challenge: 10%

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Intradermal induction: 3%
Epicutaneous induction: 15%
Challenge: 10%

No. of animals per dose:

Control group: 5 males + 5 females
Test group: 10 males + 10 females

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 physiological saline. The resulting dermal reactions were assessed 24 hours later.
Epidermal applications: Patches of filter paper (2 cm x 2 cm) were saturated with concentrations of 25%, 15%, 10% and 5% of the test article in physiological saline and applied to the clipped and shaved flanks of each of four guinea-pigs. 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. The dressings were removed after an exposure period of 24 hours and the reaction sites were assessed for erythema and edema on a numerical basis according to Draize. Further examination of the sites were performed 24 and 48 hours after removal of the dressings. Prior to the first reading, the test sites were depilated to clean of excess test article.

MAIN STUDY
A. INDUCTION EXPOSURE
- Intradermal injections:
An area of dorsal skin from the scapular region (approximately 6 cm 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 cm x 6 cm area in the clipped region as follows:
Test group:
1) Freund's complete adjuvant 50:50 with bi-distilled water.
2) The test article, diluted to 3% with physiological saline
3) The test article at the concentration used in (2), emulsified in a 50:50 mixture of Freund's complete adjuvant, and the vehicle used in (2).
Control Group:
1) Freund's complete adjuvant 50:50 with bi-distilled water.
2) Vehicle used in (2) for test group.
3) Freund's complete adjuvant 50:50 with bi-distilled water.

- Epidermal applications:
One week after the injections, the scapular area (approximately 6 cm x 8 cm) was again clipped and shaved free of hair. A 2 cm x 4 cm patch of filter paper was saturated with the test article (15% in physiological saline) and placed over the injection sites of the test animals. The patch was covered by 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 the omission of test article. Reaction sites were assessed for erythema and edema immediately, 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. Hair was clipped and shaved from a 5 cm x 5 cm area on the left and right flank of each guinea-pig. Two patches (2 cm x 2 cm) of filter paper were saturated with a) non-irritant concentration (10% in physiological saline) of the test article and b) with the vehicle only and applied to the (a) left flank and (b) right flank using the same method as for the epidermal application. The dressings were removed approximately 24 hours later. The sites were assessed for erythema and edema immediately, 24 and 48 hours after removal of the dressing, using the numerical scoring system according to Draize. The control animals were treated in the same way as described above. Prior to the first reading the test sites were depilated to clean of excess test article.

Challenge controls:

A control group (Formaldehyde-solution) is tested twice a year in the testing laboratory for sensitivity check of the guinea pig strain. The most recent test was run between November and December, 1989.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10% in physiol. saline

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10% in physiol. saline. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10% in physiol. saline

No. with + reactions:

2

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10% in physiol. saline. No with. + reactions: 2.0. Total no. in groups: 20.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10% in physiol. saline

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10% in physiol. saline. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10% in physiol. saline

No. with + reactions:

1

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% in physiol. saline. No with. + reactions: 1.0. Total no. in groups: 20.0.

The highest non-irritating concentration used for challenge application was 10%. No toxic symptoms were evident in the guinea pigs of neither the control nor test group. No death occurred.

Positive erythema reactions after first challenge procedure (due to the unequivocal findings observed after the first challenge, no second challenge was performed):

	after 24 hours		after 48 hours
	positive	total	positive	total
	% positive of total		% positive of total
CONTROL GROUP
Test substance (left flank)	0	10	0	10
	0		0
Vehicle (right flank)	0	10	0	10
	0		0
TEST GROUP
Test substance (left flank)	2	20	1	20
	10		5
Vehicle (right flank)	0	20	0	20
	0		0

Interpretation of results:

GHS criteria not met

Conclusions:

From the results of this study no allergenic potency of the test article was concluded.

Executive summary:

The study followed OECD guideline 406 (1983) and the principles of GLP. The purity of the test item is 94.6%. Each five male and female albino guinea pigs were used in control groups and each ten male and female animals in the test group. For intradermal injections of the test group, 0.1 ml of a 3% solution in PBS, Freund’s complete adjuvant (50:50) or 3% in Freud’s adjuvant was used. For challenge one week later, the highest non-irritating dose of 10% was used. One day after challenge, 2/20 showed a positive reaction for erythema compared to 0/10 in the control group. After two days, 1/20 animals showed a positive reaction compared to 0/10 in the control group.

Conclusion: From the results of this study no allergenic potency of the test article was concluded.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

All studies have been performed on read across, structural analogues.

On the bases of the reliability of matetrials available, Direct red 23 (DR23) can be evaluated as not sensitising.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Direct Red 23 is not expected to be a respiratory sensitizing

Justification for classification or non-classification

SKIN SENSITIZATION

Category 1

Substances shall be classified as skin sensitizers in category 1 where data are not sufficient for sub-categorisation in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test.

Specific criteria of animal test:

when an adjuvant type test method for skin sensitisation is used, a response of at least 30 % of the animals is considered as positive.

For a non-adjuvant Guinea pig test method a response of at least 15 % of the animals is considered positive.

Furthermore, stimulation index of three or more is considered a positive response in the local lymph node assay.

Sub-category 1A

Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.

Specific criteria:

Local lymph node assay-EC3 value ≤ 2 %

Guinea pig maximisation test-≥ 30 % responding at ≤ 0,1 % intradermal induction dose or ≥ 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose

Buehler assay - ≥ 15 % responding at ≤ 0,2 % topical induction dose or ≥ 60 % responding at > 0,2 % to ≤ 20 % topical induction dose

Sub-category 1B

Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.

Local lymph node assay - EC3 value > 2 %

Guinea pig maximisation test- ≥ 30 % to < 60 % responding at > 0,1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose.

Buehler assay - ≥ 15 % to < 60 % responding at > 0,2 % to ≤ 20 % topical induction dose or ≥ 15 % responding at > 20 % topical induction dose.

 

Based on the results obtained in the in vivo test and according to the paragraph 3.4. of the CLP Regulation n. 1272/2008, the test substance is NOT classified.