Hydrogen [4-[[4-(diethylamino)-o-tolyl][4-[ethyl(3-sulphonatobenzyl)amino]phenyl]methylene]cyclohexa-2,5-dien-1-ylidene](ethyl)(3-sulphonatobenzyl)ammonium, sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The test was conducted by means of Read Across approach. Further information was attached at section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An appropriate guinea pig maximisation test was already avaiable, which would not justify conducting an additional LLNA due to animal welfare.

Test material

Test material information

Constituent 1

Reference substance name:

Acid Blue 015 - Similar Substance 02

IUPAC Name:

Acid Blue 015 - Similar Substance 02

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: DHPW

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: no
- Age at study initiation: 5 to 8 weeks
- Weight at study initiation: medium weight of 355 g (303 to 421 g).
- Housing: 5 animal per cage (macrolon cage type IV)
- Diet (e.g. ad libitum): ad libitum (Altromin®3020)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): ca. 50 %
- Air changes (per hr): approx. 10 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light (from 6 a.m to 18 p.m) and 12 dark

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: Complete Freund adjuvant and physiological saline

Concentration / amount:

0.1 ml of test substance at 5 %

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

0.5 ml of test substance at 12 %

Day(s)/duration:

7 days after induction

Challenge

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

0.5 ml of test substance at 12 %

Day(s)/duration:

day 21 after intradermal induction

No. of animals per dose:

20 animals in the test group
10 animals in the control group

Details on study design:

INTRADERMAL INDUCTION
One day before the application, the back and flanks of the guinea pigs were shorn. Starting from behind the neck, three injections were conducted in a row on the left and right of the spinal. The distance between the injection sites was 1-2 cm; the application volume per injection site was 0.1 ml.
The animals of the three groups were treated as follows:
TEST GROUP
1. Injection sites cranial:
Complete Freund adjuvant (Difco Lab.) 1:1 with physiological saline.
2. Injection sites medial: Special Violet S-4BF 5% IG in physiolog. saline.
3. Injection place caudal: Special Violet S-4BF 5% IG in physiolog. saline and complete Freund adjuvant are formulated in equal parts.
CONTROL GROUP
The animals of the control groups were treated like the animals of the test group, but the formulations for the injection did not contain the test substance but a corresponding amount of physiolog. saline.

TOPIC INDUCTION
A week after the Intradermal induction, the topical induction took place.
On the day before, the treatment areas of the animals were shorn and treated with Paraffinol with a 10% preparation of sodium laurylsulfate. Hypoallergenic Wound plasters (2 x 4 cm) were placed between or on the injection sites, covered with aluminium foil and fixed to the skin for 48 hours with a Fermoflex self-adhesive tape (Transatlantic GmbH, Schwarzenbach).
The plasters were treated as follows:
A) test group: 0.5 ml special violet S-4BF 12% IG
b) Control groups: 0.5 ml physiolog. saline
At the end of the exposure period, the substance was removed with sterile physiolog. saline.

CHALLENGE
The challenge took place three weeks after intradermal induction.
One day before the challenge, the animals were shorn on the back and flank. During the challenge, the left flank of the animals of the test group and the first control group were placed with a 12% test substance and hypoallergenic wound plaster was fixed for 24 hours with a Fermoflex self-adhesive tape on the skin.
On the right flank, only plaster was fixed for comparison.
The application volume was 0.5 ml each. At the end of the exposure time, the skin of the animals was shaved in the area of the treatment with Pilca cream (Olivein, Hamburg).

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

12 % of test substance

No. with + reactions:

0

Total no. in group:

20

Results 2

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

12 % of test substance

No. with + reactions:

0

Total no. in group:

20

Any other information on results incl. tables

General Investigations

The treatment was tolerated by all animals with no symptoms. Death did not occur.

Changes in the body weight of the animals in the treatment group corresponded to that of the control group.

Testing on Skin sensitising effect

There were no skin reactions in test group and control animals.

Discussion and evaluation

the test substasnce was studied at guinea pigs in a maximization test after MAGNUSSON and KLIGMAN on skin sensitizing properties. The Intradermal induction was made with a 5%, the topical with a 12% test substance conducted. Higher concentrations than 12% could not be formulated. In the dose determination attempt to determine the induction concentration, the 12% formulation proved to be non-irritating to the skin; The treatment fields were therefore pre-irritated with sodium laurylsulfat before topical induction. A 12% formulation was used in the provocation.

After exposure to provocation and dose determination, the animals were chemically dehaired in the area of the treatment fields. In this way, the oberflachlichen, corneal skin cells also remove a Grosteil of the dye and thus facilitate the assessment of the Entzundlichen Hautrotung.

After the provocation, skin reactions did not occur in the control or in the PrUfmustergruppe.

Special Violet S-4BF therefore has no skin sensitizing potential.

Applicant's summary and conclusion

Interpretation of results:

other: CLP criteria not met

Conclusions:

Not skin sensitising

Executive summary:

Method

The substance was tested for its skin sensitisation potential according to the method described in the OECD guideline 406, in male guinea pigs. 20 animals were used in the test group and 10 in the control group.

The intradermal induction was performed at 5 % of test substance formulation while the topical induction and the challenge were performed at 12 %.

After challenge skin reaction did not occur in none of the animal of the test and control group.

Conclusion

Not skin sensitizer