Sodium 1-amino-4-[[3-[(benzoylamino)methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Endpoint summary

Description of key information

Non sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 15 to April 11, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

yes

Remarks:

in the intradermal induction a mixture of FCA: physiological saline was chosen instead of FCA

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.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: WIGA
- Age at study initiation: 7 - 9 weeks at beginning of acclimatization period.
- Weight at study initiation: control and test group 398 - 506 g; Pretest 439 - 512 g.
- 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 animal 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.
- Acclimation period: one week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C
- Humidity (%): between 45 - 67%
- 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.

Route:

intradermal and epicutaneous

Vehicle:

other: (Pretest) Intradermal: bidistilled water; Epidermal: vaselinum album; (Induction) Intradermal: bidistilled water, 1:1(v/v) mixture of Freund's complete adjuvant and physiological saline ; Epidermal: vaselinum album;

Concentration / amount:

5% (intradermal injection)
25% (epidermal application)

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: vaselinum album.

Concentration / amount:

15 %

No. of animals per dose:

20

Details on study design:

RANGE FINDING TESTS: yes.

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 5% 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. 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 blue 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 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 15% was selected.

MAIN STUDY
1. INDUCTION EXPOSURE

A. 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.
Control 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 a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

B. 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) was clipped, shaved free of hair and the test area was pretreated with 10 % Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum. 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. One test day 8 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. 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.

2. 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 ( 2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 15% (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. 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.

Challenge controls:

Yes. The right flank was treated with the vehicle alone.

Positive control substance(s):

yes

Remarks:

4-Aminobenzoic Acid Ethyl Ester.

Positive control results:

According to the procedures used in this experiment positive results were observed in the treated animals after the epidermal challenge application.
In this study 30-50% of the animals were positive after treatment with a non-irritant test substance concentration of 25% and with concentrations of 10% and 5% in mineral oil. Therefore according to EEC (European Economic Community) classification criteria described in guidelines 93/21/EEC (EEC Official Journal Nr. L 110A, May 04, 1993), this test article is considered to be a sensitizer.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

15% test substance

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

one animal of the test group was found dead on test day 4

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

15% test substance

No. with + reactions:

0

Total no. in group:

10

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

25% 4-aminobenzoic acid ethyl ester

No. with + reactions:

10

Total no. in group:

20

Reading:

rechallenge

Hours after challenge:

24

Group:

positive control

Dose level:

10% 4-aminobenzoic acid ethyl ester

No. with + reactions:

8

Total no. in group:

20

Reading:

rechallenge

Hours after challenge:

24

Group:

positive control

Dose level:

5% 4-aminobenzoic acid ethyl ester

No. with + reactions:

6

Total no. in group:

20

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

15% test substance

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

one animal of the test group was found dead on test day 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

15% test substance

No. with + reactions:

0

Total no. in group:

10

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

25% 4-aminobenzoic acid ethyl ester

No. with + reactions:

9

Total no. in group:

20

Reading:

rechallenge

Hours after challenge:

48

Group:

positive control

Dose level:

10% 4-aminobenzoic acid ethyl ester

No. with + reactions:

7

Total no. in group:

20

Reading:

rechallenge

Hours after challenge:

48

Group:

positive control

Dose level:

5% 4-aminobenzoic acid ethyl ester

No. with + reactions:

6

Total no. in group:

20

MAIN STUDY

SKIN EFFECTS AFTER INTRADERMAL INDUCTION PERFORMED ON TEST DAY 1

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 6, followed respectively by necrosis, encrustation and exfoliation of the encrustation up to the termination of test.

Injection site 2 (bi-distilled water) The area around the injection site was oedematous and erythematous from test day 2 to 6.

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 (5% solution of test article in bi-distilled water) The area around the injection site was oedematous and blue discolored from test day 2 to 6, and 2 to 8 respectively, followed by necrosis, encrustation and exfoliation of the encrustation up to the termination of test.

Injection site 3 (5% 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 injection site 2 with the 5% 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 vaselium album only. TEST GROUP: As the test article stained the skin blue, it was not possible to determine whether erythema was present. However, no oedema was observed. Blue discolouration was noted from test day 10 to 28.

All the animals were pretreated with SLS in paraffinum perliquidum.

 

SKIN EFFECTS AFTER THE CHALLENGE PERFORMED ON TEST DAY 22

CONTROL AND TEST GROUP:

Challenge: no positive reactions were observed in the animals either when treated with vaselinum album alone or when treated with the test article at 15% in vaselinum album.

 

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS:

One animal of the test group was found dead on test day 4. At necropsy gray white foci (D=10mm) were observed on the heart, the lungs were not collapsed with several dark red foci (D=10mm) and the spleen was enlarged (D=40x23mm) with the presence of gray white, firm nodules (D=5mm). One animal of the epidermal pretest was found dead on test day 7. At necropsy the lungs were not collapsed with several dark red foci (D=5mm) and the right cranial lobe was dark red discoloured.

 

CLINICAL SIGNS, SYSTEMIC:

No symptoms of systemic toxicity were observed in the animals.

 

BODY WEIGHTS:

The body weight of the animals was within the normal range of variability.

Interpretation of results:

GHS 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. Ten males were used as control group and 20 males were used as test group as described in the OECD guideline 406.

 

Results:

The highest non-irritating test article concentration used for challenge application was 15% in vaselinum album.

 

Conclusion:

0% of the animals were positive after treatment with the highest non-irritant test substance concentration of 15% in vaselinum album. Therefore the test article considered to be a non sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The potential of the test item to cause skin sensitisation reactions following topical application to the skin of albino guinea pigs, was assessed using the Maximization Test of B. Magnusson and A.M. Kligman (1969), according to the OECD Guideline for testing of chemicals No. 406.

0% of the animals were positive after treatment with the highest non-irritant test substance concentration of 15% in vaselinum album.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

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 animal test (Guinea pig maximisation test) results performed, according to the paragraph 3.4. of the CLP Regulation n. 1272/2008, the test substance is Not Classified, as 0% of the animals were positive after treatment (intradermal dose = 5% of test substance).