Tetrasodium 2,2''-diamino-8,8''-dihydroxy-4',4'''-{4-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,6-diyldiimino}bis(naphthalene-1-azobenzene-2',6-disulfonate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 18th to July 26th, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

a guinea pig maximisation test is already available

Test material

Test material information

Constituent 1

Reference substance name:

Red LF 6339

IUPAC Name:

Red LF 6339

In vivo test system

Test animals

Species:

guinea pig

Strain:

Himalayan

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd, Biotechnology and Animal breeding Division, Wolferstrasse 4, CH-4414, Fullinsdorf
- Age at study initiation: 3-6 weeks
- Weight at study initiation: 385-420 g (pre-test), 304-437 g (beginning of acclimatisation period)
- Housing: individually in Makrolon type-4 cages with standard softwood bedding
- Diet (e.g. ad libitum): pelleted standard Provimi Kliba 3418 guinea pig breeding/maintenance diet containing vitamin C, ad libitum
- Water (e.g. ad libitum): community tap water, ad libitum
- Acclimation period: one week under test conditions after health examination. No acclimatization for the animals of the pretest

ENVIRONMENTAL CONDITIONS
- Temperature: 20±3 °C
- Humidity: 30-70 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Concentration / amount:

5 % (intradermal)
50 % (epidermal)

Day(s)/duration:

day 1 (intradermal); day 8 (epidermal)

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Concentration / amount:

10 %

Day(s)/duration:

Day 22

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

15 (10 test and 5 control)

Details on study design:

VEHICLE
Polyethylene glycol (PEG 300)

TEST ITEM PREPARATION: the test item and vehicle or auxilliary compound were placed into a glass beaker and a weight by weight dilution was prepared.

SELECTION OF CONCENTRATION OF TEST ITEM FOR MAIN TEST
- Intradermal induction: the 5 % concentration of test item used for the intradermal induction exposure was well-tolerated systemically and was the highest technically applicable concentration
- Epidermal Induction: the 50 % concentration of test item used for the epidermal induction exposure was well-tolerated systemically and was the highest technically applicable concentation causing milk skin irritation
- Epidermal challenge: the 10 % concentration of the test item used for the challenge application was the maximum tested non-irritant concentration.

PRETEST:
- Intradermal injections: four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freud's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of 5 %, 3 % and 1 % of the test item in PEG 300. Dermal reactions were assessed 24 hours later. The test item concentration of 5 % was selected for intradermal induction in the main study.
- Epidermal applications: four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freud's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper were saturated with the test item at 50 %, 25 %, 15 % and 10 % in PEG 300 and applied to the clipped and shaved flanks. The patches were covered by a strip of aluminium foil and firmly secured by elastic plaster wrapped around the trunk and covered with imprevious adhesive tape. The dressings were removed after an exposure period of 24 hours. Twenty-one hours after removal of the dressing, the applicatio site was depilated with an approved depilatory cream in order to visualize any resulting erythema. The depilatory cream was placed on the patch sites and surrounding areas and left on for 3-5 minutes. It was then thoroughly washed off with a steam of warm, water. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema.

MAIN STUDY
A. INDUCTION EXPOSURE
A.1 INTRADERMAL INJECTIONS
- No. of exposures: 3 pairs of intradermal injections
- Test groups: 1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline, 2) the test item at 5 % in PEG 300, 3) the test item at 5 % 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) PEG 300, 3) 1:1 (v/v) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
- Site: area of dorsal skin from the scapular region

A.1 EPIDERMAL APPLICATIONS
- Exposure: one week after the injections
- Test groups: a patch of filter paper was saturated with the test item (50 % in PEG 300) and placed over the injection sites of the test animals. The patch was covered for an ensured intensive contact of the test item.
- Control group: treated as the test group but only with PEG 300.
- Site: area of dorsal skin from the scapular region
- Evaluation of the sites: 24 and 48 hours after removal of the bandage for erythema and oedema.

B. CHALLENGE EXPOSURE
- Day(s) of challenge: two weeks after the epidermal induction application.
- Exposure period: 24 hours
- Test groups and control groups: two patches of filter paper were saturated with the test item (10 % in PEG 300) applied to the left flank and the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The patch was covered for an ensured intensive contact of the test item.
- Site: left and right flank of each guinea pig
- Concentrations: 10 %
- Evaluation (hr after challenge): 24 and 48 hours after removal of the bandage for erythem and oedema according to the method of Magnusson and Kligman.

OBSERVATIONS
- Viability/Mortality: daily from delivery of the animals to the termination of the test
- Clinical signs: daily from delivery of the animals to the termination of the test
- Skin reactions: at the times specified durinh the pretest, induction and challenge periods
- Body weights: at pretest and acclimatization start, day 1 and termination of the test

Positive control substance(s):

yes

Remarks:

alpha-hexylcinnamaldehyde

Results and discussion

Positive control results:

100 % and 60 % positive responses at 24 and 48 h readings in test group.

In vivo (non-LLNA)

Results

Results 1

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

test item in 10 % in PEG 300 (left flank) and PEG 300 only (right flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no clinical signs and no mortality

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

test item in 10 % in PEG 300 (left flank) and PEG 300 only (right flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no clinical signs and no mortality

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

induction at 10 % (intradermal and epidermal); challenge at 0.1 % (epidermal)

No. with + reactions:

6

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

- Intradermal induction: erythema, oedema, necrotizing dermatitis, encrustration, exfoliation of encrustation and violet discoloration

- Epidermal induction: control group- no erythematous or oedematous reaction was observed in the animals treated with PEG 300 only. Test group- the test item stained the skin violet and it was not possible to determine whether erythema was present or no. No oedema was observed. The animals were not depilated in the epidermal induction phase.

- Challenge: control group- no skin reactions in the animals treatd with either PEG 300 only or when treated with the test item at 10 % in PEG 300. Violet discoloration produced by the test item was noted directly after removal of the patch. Test group-  no skin reactions in the animals treatd with either PEG 300 only or when treated with the test item at 10 % in PEG 300. Violet discoloration produced by the test item was noted directly after removal of the patch.

Applicant's summary and conclusion

Interpretation of results:

other: not classified as a skin sensitiser according to the CLP Regulation (EC) No.1272/2008

Conclusions:

not skin sensitiser