Tetra-sodium/lithium 4,4'-bis-(8-amino-3,6-disulfonato-1-naphthol-2-ylazo)-3-methylazobenzene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 16, 1990 - March 2, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD 406 Guideline study, GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

Tetra-sodium/lithium 4,4'-bis-(8-amino-3,6-disulfonato-1-naphthol-2-ylazo)-3-methylazobenzene

EC Number:

408-210-8

EC Name:

Tetra-sodium/lithium 4,4'-bis-(8-amino-3,6-disulfonato-1-naphthol-2-ylazo)-3-methylazobenzene

Cas Number:

124605-82-9

Molecular formula:

C33 H26 N8 O14 S4 .x Li .x Na

IUPAC Name:

dilithium(1+) disodium 5-amino-3-{2-[4-(2-{4-[2-(8-amino-1-hydroxy-3,6-disulfonatonaphthalen-2-yl)diazen-1-yl]-2-methylphenyl}diazen-1-yl)phenyl]diazen-1-yl}-4-hydroxynaphthalene-2,7-disulfonate

Details on test material:

- Physical state: powder
- Stability under test conditions: stable for at least 2 hours

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Himalayan spotted

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., 4414 Fullinsdorf
- Age at study initiation: males : 7 weeks, females: 8 weeks
- Weight at study initiation: males: 348 - 399 g, females: 315 - 375 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard Kliba 342, Batches 55/89 and 56/90 guinea pig breeding/ maintenance diet ("Kliba", Klingentalmuhle AG, 4303 Kaiseraugst), ad libitum.
- Water: Community tap water from Itingen, ad libitum.
- Acclimation period: one week under test conditions after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20+/-3 °C
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 air changes per hour

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

physiological saline

Induction 2

Concentration / amount:

Intradermal induction: 5 % in physiological saline;
Epidermal induction: 15 % in physiological saline;
Challenge: 10 % in physiological saline;

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Challenge 2

Concentration / amount:

Intradermal induction: 5 % in physiological saline;
Epidermal induction: 15 % in physiological saline;
Challenge: 10 % in physiological saline;

No. of animals per dose:

control group: 10
test group: 20

Details on study design:

RANGE FINDING TEST:
The objective of this investigation was to identify irritant test article concentrations suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application.
The procedure employed for these investigations was as follows:

- 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 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. This procedure ensured the intensive contact of the test article. 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 the scale described above. Further examination of the sites were performed 24 and 48 hours after removal of the dressings. Prior to the readings the test sites were depilated.

MAIN STUDY
Induction
Intradermal injections: 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) Freund's complete adjuvant 50:50 with bi-distilled water.
2) The test article, diluted to 5% 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 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 (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 described previously.

Challenge
The test and control guinea-pigs were challenged two weeks after the epidermal induction application. Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea-pig. Two patches ( 2 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 as described under preliminary study. The control animals were treated in the same way as described above. Prior to the first reading the test sites were depilated.

Re-challenge
A second challenge was performed two weeks after the first challenge. The treatment procedure for the animals of the test group was similar as described for the first challenge with the exception that the flanks of all the guinea-pigs were changed (a - vehicle; b - test article dilution). The control animals were treated with the vehicle alone on the left flank. Prior to the first reading the test sites were depilated.

Positive control substance(s):

yes

Remarks:

A control group (Formaldehyde-solution) is tested twice a year for sensitivity check of the guinea pig strain

Study design: in vivo (LLNA)

Statistics:

Mean values with standard deviations.
Fisher-Test (The Exact Fisher Test for comparison of the basic probability of two binominal distributions. L. Sachs, Statistische Auswertungsmethoden, Georg Thieme Verlag, Stuttgart 1971).
For calculation of p-values the 24-hour reading of the animals from the control and test group was used.

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10 %

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 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10 %

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 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10 %

No. with + reactions:

6

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 6.0. Total no. in groups: 20.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10 %

No. with + reactions:

4

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 %. No with. + reactions: 4.0. Total no. in groups: 20.0.

Results 5

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

10 %

No. with + reactions:

4

Total no. in group:

20

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 4.0. Total no. in groups: 20.0.

Results 6

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

15 %

No. with + reactions:

9

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 15 %. No with. + reactions: 9.0. Total no. in groups: 10.0.

Any other information on results incl. tables

SENSITIZING EFFECTS

CONTROL GROUP:

No erythema was found after the first challenge in response to treatment with physiological saline or to a 10% test article dilution.

No erythema was observed after the second challenge in response to treatment with physiological alone.

TEST GROUP:

First Challenge: At 24 and 48-hour readings, erythema were found in 6 out of 20 and 4 out of 20 animals, respectively, in response to a 10% test article dilution. No erythema was found in response to treatment with physiological saline alone.

Second Challenge: At 24-hour reading, erythemas were found in 2 out of 20 animals in response to a 10% test article dilution. No erythemas were found when treated with physiological saline alone.

SYMPTOMS, LOCAL

CONTROL GROUP:

Application area around the injection sites 1 and 3 was found to show erythema and edema from day 2 to 5; necroses from day 6 to 18; desiccation from day 19 to 20 and exfoliation from day 19 to 39 (termination of the study). Application area around the injection site 2 was found to show erythema and edema from day 2 to 5; necroses at day 6 and from day 10 to 18; desiccation from day 19 to 20; exfoliation from day 19 to 24 and 33 to 39 (termination of the study). In addition first challenge application area was found to show staining from day 23 to 25.

 

TEST GROUP:

Application area around the injection site 1 was found to show erythema and edema from day 2 to 5. Around the injection sites 2 and 3 edema and staining were observed from days 2 to 6; For the three application areas, necroses were observed from day 6 to 18; desiccation on days 19 and 20 and exfoliation from day 19 to 39 (termination of the study). In addition, epidermal induction-,

first challenge- and second challenge- application areas were found to show staining from day 10 to 20, 23 to 25 and 37 to 39, respectively. On day 9 of test no observation could be performed because the animals were treated semi-occlusively.

 

SYMPTOMS, SYSTEMIC

On days 1 to 3 the test animals and their veins showed bluish discoloration. Bluish discoloration was also observed in the urine.

 

BODY WEIGHTS

The body weight gain of the animals was not affected adversely during the study.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

Based on the results of this study the test article is regarded as a skin sensitizer.

Executive summary:

In a guinea pig maximization test according to OECD guideline No. 406, 10 male and 10 female animals were first induced and then challenged with the test article to investigate its sensitization potential. Induction was a two-stage operation: First, three pairs of intradermal injections were made into the clipped dorsal area of the animals with (1) Freund's complete adjuvant 50:50 with bi-distilled water, (2) the test article diluted to 5% with physiological saline, and (3) the test article at 5% in a 50:50 mixture of Freund's complete adjuvant and physiological saline. One week later, the test article in physiological saline at a concentration of 15% was applied on a filter paper patch to the scapular area of the animals (occlusive administration for 48 hours). Two weeks after the epidermal induction application the animals were tested on the right and left flank with 10 % test substance in physiological saline and the vehicle alone (24 h occlusive application). The test sites were assessed for erythema and edema immediately, 24 and 48 hours after removal of the dressing. A control group (5 m/5 f) was treated with adjuvant and the vehicle during the induction period. During the challenge period the group was treated with the vehicle as well as with the test compound. A second challenge was performed two weeks after the first challenge. The treatment procedure for the animals of the test group was similar as described for the first challenge. The control animals were treated with the vehicle alone on the left flank. After the first challenge, erythema were found in 6 out of 20 (24h reading) and 4 out of 20 animals (48 h reading) in response to treatment with the test article. No erythema reactions were found in response to treatment with physiological saline alone. At the 24-hour reading after the second challenge, erythema reactions were found in 2 out of 20 animals in response to a 10% test article dilution. No erythema reactions were found when treated with physiological saline alone. Under the experimental conditions employed, 30% of the animals of the test group showed skin reactions 24 after the first challenge treatment. Therefore, under the experimental conditions of this study the test substance is classified as a skin sensitizer.