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Toxicological information

Skin sensitisation

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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:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Adopted July 17, 1992 (reported París 29 April 1993).
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
July 31, 1992
GLP compliance:
yes (incl. QA statement)
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

Constituent 1
Chemical structure
Reference substance name:
Pentasodium bis{7-[4-(1-butyl-5-cyano-1,2-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)phenylsulfonylamino]-5'-nitro-3,3'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato} chromate (III)
EC Number:
419-210-2
EC Name:
Pentasodium bis{7-[4-(1-butyl-5-cyano-1,2-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)phenylsulfonylamino]-5'-nitro-3,3'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato} chromate (III)
Cas Number:
178452-71-6
Molecular formula:
C66H48CrN16Na5O28S6
IUPAC Name:
chromium(3+) pentasodium bis(7-{4-[2-(1-butyl-5-cyano-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridin-3-yl)diazen-1-yl]benzenesulfonamido}-2-[2-(5-nitro-2-oxido-3-sulfonatophenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate)

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Himalayan spotted
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
-Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, 4414 Füllinsdorf/Switzerland
-Females nulliparous and non-pregnant:yes
-Age at study initiation: 5 - 7 weeks
-Weight at study initiation: 402 - 466 g
-Housing: individually in Makrolon type-3 cages with autoclaved standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
-Diet: pelleted standard Kliba 342, Batch no. 66/94 (from 28-DEC-1995) and 67/94 (from 28-JAN-1994) guinea pig breeding/maintenance diet ("Kliba", KlingentalmOhle AG, CH-4303 Kaiseraugst), ad libitum.
-Water: community tap water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/l) via the drinking water
-Acclimation period: one week for the control and test groups, whereas no acclimatlzation 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: 20 - 22 °C
-Humidity: 51 - 68 %
-Air changes: 10 - 15 air changes per hour
-Photoperiod: 12 hours cycle dark/light with artificial fluorescent light (approx.100 Lux)
-Other: music during the light period.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Concentration / amount:
0 %
Day(s)/duration:
single injection
Route:
intradermal
Vehicle:
water
Concentration / amount:
5 %
Day(s)/duration:
single injection
Route:
intradermal
Vehicle:
other: emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
Concentration / amount:
5 %
Day(s)/duration:
single injection
Route:
epicutaneous, semiocclusive
Vehicle:
other: vaselinum album
Concentration / amount:
25 %
Day(s)/duration:
48 hours
Challenge
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
other: vaselinum album
Concentration / amount:
3 %
Day(s)/duration:
24 hours
No. of animals per dose:
-Intracutaneous Pretest: 2
-Epidermal Pretest I: 4
-Epidermal pretest II: 4
-Control Group: 10
-Test Group: 20
Details on study design:
PRETEST
The objective of this investigation was to identify a maximally tolerated concentration of the test item suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test item, by the topical route of administration, was identified for the challenge application.
INDUCTION EXPOSURE: INTRADERMAL INJECTION
-No. of exposures: 1
-Test groups: 2 guinea pigs
-Site: clipped flank
-Frequency of applications: single injection
-Concentrations: 0.1 ml/site of 5, 3 and 1% of the test item in bi-distilled water.
-Evaluation: the resulting dermal reactions were assessed 24 hours later.
INDUCTION EXPOSURE: EPIDERMAL APPLICATION
-No. of exposures: 4 applications of filter paper ( 2 x 2 cm) saturated with test item
-Exposure period: 24 hours
-Test groups: 4 guinea pigs
-Site: clipped and shaved flanks
-Protection site: the patches were covered by a strip of aluminium 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 item.
-Concentrations in vaselium album:
A = 25 %
B = 15 %
C = 10 %
D = 5 %
-Evaluation: the reaction sites were assessed 24 and 47 hours after removal of the bandage for erythema and oedema.
INDUCTION EXPOSURE: EPIDERMAL APPLICATION II (to determined the highest non-irritating concentration)
-No. of exposures: 4 applications of filter paper ( 2 x 2 cm) saturated with test item
-Exposure period: 24 hours
-Test groups: 4 guinea pigs
-Site: shaved flanks
-Protection site: the patches were covered by a strip of aluminium 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 item.
-Concentrations in vaselinum album:
A = 3 %
B = 1 %
C = 0.5 %
D = 0.3 %
-Evaluation: the test sites were depilated approximately 21 hours after removing of the dressing.

MAIN STUDY
A1. INDUCTION EXPOSURE: INTRADERMAL INJECTIONS (performed on test day 1)
-No. of exposures: three pairs of injections (0.1 ml/site)
-Exposure period: single injection
-Test groups: 20 female guinea pigs
-Control group: 10 female guinea pigs
-Site: scapular region (4 x 6 cm) in the clipped region
-Frequency of applications: one application for each site
-Concentrations:
test group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test item diluted to 5% with bi-distilled water
3) The test item 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 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

A2. INDUCTION EXPOSURE: EPIDERMAL APPLICATION (performed on test day 8)
-No. of exposures: one application with 2 x 4 cm patch of filter paper was saturated with the test item.
-Test groups: 20 female guinea pigs
-Control group: 10 female guinea pigs
-Site: scapular area (6 x 8 cm) in clipped and shaved region (over the injection site)
-Protection site: the patch was covered with aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape.
-Frequency of applications: one application
-Duration: for 48 hours
-Concentrations:
test group:0.5 ml of a 25% (w/w) test substance concentration
control group: vaselinum album only
-Evaluation: the reactiont sites were assesed for erythema and oedema 24 and 48 hours after removal of the dressing.

B. CHALLENGE EXPOSURE (performed on day 22)
-No. of exposures: 2 patches ( 2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration (left flank) and the vehicle only (vaselinum album, applied to the right flank) using the same method as for the epidermal application.
-Exposure period: 24 hours
-Test groups: 20 female guinea pigs
-Control group: 10 female gunea pigs
-Site: flanks ( 5 x 5 cm) area in clipped and shaved region
-Concentrations:
test group
3 %
control group
vehicle only (vaselinum album)
-Evaluation: 24 and 48 hours after removal of the dressings.
Challenge controls:
yes
Positive control substance(s):
yes
Remarks:
4-aminobenzoic acid ethyl ester and 2-mercaptobenzothiazol (tested in two current reference studies)

Results and discussion

Positive control results:
2-mercaptobenzothiazol
No toxic symptoms were evident in the guinea pigs of the control or test group. The 60% of the animals were positive after treatment with a non-irritant test substance concentration of 25% in mineral oil.

4-aminobenzoic acid ethyl ester
No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred. In this study 60% of the animals were positive after treatment with a non-irritant test substance concentration of 25% in mineral oil.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
vaselinum album only (right flank) and 3 % in vaselinum album (left flank)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vaselinum album only (right flank) and 3 % in vaselinum album (left flank)
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vaselinum album only (right flank) and 3 % in vaselinum album (left flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vaselinum album only (right flank) and 3 % in vaselinum album (left flank)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25 % of 2 -mercaptobenzothiazol in mineral oil
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25 % of 2 -mercaptobenzothiazol in mineral oil
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25 % of 4-aminobenzoic acid ethyl ester in mineral oil
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25 % of 4-aminobenzoic acid ethyl ester in mineral oil
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

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 5, and became necrotic from test day 6 to 7. Encrustation was observed from test day 8 to 15 and exfoliation of encrustation from test day 16 to the termination of test.

-Injection site 2 (bi-distilled water): no local symptoms were observed.

-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 % dilution of test article in bi-distilled water) : the area around the injection site was oedematous and green discoloured from test day 2 to 5 and 2 to 10 respectively, necrotic from test day 6 to 7. Encrustation was observed from test day 8 to 15 and exfoliation of encrustation from test day 16 to the termination of test.

-Injection site 3 (5% dilution of test article in a 1:1 (v/v) mixture of FCA and physiological saline): the area around the injection site was oedematous and green discoloured from test day 2 to 5 and 2 to 13 respectively, necrotic from test day 6 to 7. Encrustation was observed from test day 8 to 15 and exfoliation of encrustation from test day 16 to the termination of test.

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 dark green, it was not possible to determine whether erythema was present. However, no oedema was observed. Green discolouration was noted from test day 11 to 27 (termination of test).

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 3% in vaselinum album. Green discolouration was noted from test day 23 (after removal of the dressing) to 24 (prior to the depilation).

 

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

One animal of the control group, one of the test group, one of the intradermal pretest, three animals of the epidermal pretest I and all animals of the epidermal pretest II lost weight during the acclimatization period. The loss of weight is considered to be of incidental nature.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified according to the CLP Regulation (EC 1272/2008)
Conclusions:
The substancewas found not be sensitiser under the test conditions.
Executive summary:

In order to assess the cutaneous allergenic potential of test substance, the Maximization-Test was performed in 30 (10 control and 20 test) female albino guinea pigs, in accordance with OECD Guideline No. 406 (1992) and the method B.6 of EEC-Directive 96/54 EEC. Three pre-tests were performed in order to identify the maximally tolerated concentration of the test substance suitable for the induction phase and the suitable non-irritant concentration of the test substance for the challenge application in the main study. For intradermal and epidermal induction, the test item concentrations selected were 5 % in water and 25 % in vaselium album respectively. The highest non-irritating test item concentration used for challenge application was 3% in vaselinum album.

No toxic symptoms were evident in the guinea pigs of the control or test group and no deaths occurred. None of the animals of the test and control groups were observed with positive skin reactions after treatment with the selected non-irritant test substance concentration of the challenge, at both readings (24 and 48 hours).

The test substance is considered to be a non-sensitizer under the test conditions.