2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium methyl sulphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-07-17 to 1995-09-14

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 guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

Existing data from 1995

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium chloride

EC Number:

248-053-0

EC Name:

2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium chloride

Cas Number:

26850-47-5

Molecular formula:

C19H23ClN4O2S

IUPAC Name:

2-[(E)-{4-[Ethyl(2-hydroxyethyl)amino]phenyl}diazenyl]-6-methoxy-3-methyl-1,3-benzothiazol-3-ium chloride

Test material form:

solid

Details on test material:

see below

In vivo test system

Test animals

Species:

guinea pig

Strain:

Himalayan

Sex:

male

Details on test animals and environmental conditions:

Test Animals:
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, CH-4414 Füllinsdorf/Switzerland
- Age at arrival: 5-7 weeks old
- Weight at arrival: 368-498 grams
- Housing: Individually in Makrolon type-3 cages (size: 22x37x15 cm) with autoclaved standard softwood bedding S 8/15, batch 231 (“Lignocel, Schill AG, CH-4132 Muttenz)
- Diet (e.g. ad libitum): Ad libitum pelleted standard Kliba 342, Batch no. 69/95 guinea pig breeding/maintenance diet (“Kliba”, Klingentalmühle AG, CH-4303 Kaiseraugst)
- Water (e.g. ad libitum): Ad libitum Comunitx tap-water from FDüllinsdorf. Once weekly additionally supply of ascorbic acid (1g/l).
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Humidity (%): 52 – 86 %
- Air changes (per hr): Approximately 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12, music during light period

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

Each 25 mm Hill Top Chamber was saturated by the freshly prepared test substance solution (50 % in bidistilled water), applied with a spatula. Dose applied wasnot determined.

Day(s)/duration:

three weekly 6-hour applications

Adequacy of induction:

other: highest concentration of induction screen (50 %) showed no adverse effect.

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50% in bidistilled water

Day(s)/duration:

6 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

20 guinea pigs in the test group, 10 in the control group

Details on study design:

Treatment Methods:
Patching method: The same patching method was used for induction, irritation screens and challenge. The animals’ fur was shaved on the day before exposure with a fine clipper blade. Closed patches were applied to the animals as follow:
Each 25 mm Hill top Chamber was saturated by freshly prepared substance solution, applied with a spatula. The dose applied was not determined. The animals were put in the restrainer and the designated patch was applied to the clipped surface as quickly as possible after the test item has been applied. The patch appliance was occluded with a rubber dental dam. The rubber dental gum used for occlusion of the patches was of medium gauge 12-15 cm wide, depending on the animal size and the number of patches to be covered. The rubber dental dam was pulled snug on each side of the animal and secured with one more large size clip on each side of the restrainer. The rubber dental dam was placed under the front and back metal restraining bands and had snug contact with the animal over the entire dorsal surface. The restrainers were adjusted to minimize movement of the animals during the exposure period. Six hours later, the rubber dental dams and patches were removed and the animals were taken away from the restrainers. After the animals were returned to their individuals cages.

Induction:
The inductions phase of sensitization testing consisted of three weekly 6-hours applications of the test substance at the same site on the left shaved shoulder of the test animal.
The induction was performed with the test article at 50% in bidistilled water. After the last induction exposure, the animals were left untreated for 14 days before primary challenge. The responses were graded 24 hours (± 2 hours) after patch removal, according to the grading method described below. Any gross skin reactions were recorded without depilation.

Challenge:
The animals previously exposed during the induction period (i.e. test group) as well as the previously untreated control animals were challenged two weeks after the last induction exposure using the test article concertation of 50 % in bidistilled water. The fur was clipped from the left posterior quadrant of the side and back of the animals. Patch sites for challenge are indicated below. The exposure period was 6 hours on a naïve skin site. The responses were graded at 24 and 48 hours after the patches have been removed according to the grading method described below.

Grading method:
The animals used for irritation screens and challenge were depilated eighteen to twenty-two (18-22) hours after patches had been removed, using an approved depilatory cream (VEET cream, Reckitt & Colman AG, CH-4123 Allschwil). 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 dried with a disposable towel, and returned to their cages.
Erythema and oedema were assessed as follows:
Erythema:
0 = no erythema
± = slight, patchy erythema (i.e. barely perceptible or questionable reaction)
1 = slight confluent erythema (i.e. a slight but definite reaction at the patch site) or moderate, but patchy erythema (i.e. moderate erythema involving at least 50% or more of the area of the patch site.)
2= moderate confluent erythema
3= severe erythema
Oedema (according to Draize):
0 = no oedema
1 = very slight oedema (barely perceptible)
2 = well-defined oedema (edges of area well-defined by definite raising)
3= moderate oedema (raised approx. 1mm)
4= severe oedema (raised more than 1 mm and extending beyond the area of exposure)
The grading method used for irritation screens, induction and challenge was identical. This one was performed 24 hours (± 2 hours) after removal of the patches for irritation screens, induction and challenge and repeated 24 hours (± 2 hours) later (48-hour grades) for irritation screen and challenge.

OTHER:
- Clinical signs of toxicity: yes, mortality and clinical signs were checked
- Body weight: yes, body weight measurement were conducted

Challenge controls:

10 animals previously untreated were challenged together with the test group using the test article concentration of 50 % in bi-distilled water.

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 % in bi-distilled water

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

2 animals with slight confluent erythema

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 % in bi-distilled water

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

2 animals with slight patchy erythema

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no effects

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no effects

Remarks on result:

no indication of skin sensitisation

Results 5

Group:

positive control

Remarks on result:

not measured/tested

Any other information on results incl. tables

Table 1: Results of the induction screening

Irritancy results
	24 hours reading Concentrations (%) of the test substance				24 hours reading Concentrations (%) of the test substance
Response grade (erythema)	50	25	15	10	50	25	15	10
0	4*	4	4	4	4	4	4	4
±	0	0	0	0	0	0	0	0
1	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0

 * = number of animals showing response

Grade 0 and ± are considered to be representative of insignificant response, whereas those of one or greater are considered to be significant. The results of the 24- and 48 hours reading were taken into consideration and in this case the test article at 50, 25, 15, and 10 % resulted in 4 scores of 0. Therefore the most representative concentration to stimulate a state of immune hypersensitivity is 50% in bi-distilled water.

Table 2 : Results of the challenge screening

	24 hours reading Concentrations (%) of the test substance				24 hours reading Concentrations (%) of the test substance
Response grade (erythema)	50	25	15	10	50	25	15	10
0	0*	0	0	0	0	0	0	0
±	0	0	0	0	0	0	0	0
1	0	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0	0

 * = number of animals showing response

The highest non-irritating concentration is determined as the concentration which induces responses in the four guinea pigs no more severe than two grades of +/-and two of 0. In this case, all concentrations resulted in 4 scores of 0. Therefore, the highest non-irritating concentration for the challenge was determined as the concentration of 50% in bi-distilled water

 

Table 3: Primary Sensitization Results (Incidence Tables)

Erythema score	Test group 20 animals		Control group 10 animals
	24 hrs	48 hrs	24 hrs	48 hrs
0	18	18	10	10
±	0	2	0	0
1	2	0	0	0
2	0	0	0	0
3	0	0	0	0
No. with grades ≥ 1	2	0	0	0
No. Tested	20	20	20	20
Total**	2		0

** The Total is the number of animals showing a grade ≥ 1 at either 24 or 48 hours.

10% of the animals of the test group were observed with positive reactions (grade 0 and ± are considered to be non-significant responses, whereas those of 1 and greater are considered to be significant) after challenge performed with the highest non-irritating concentration of the test item at 50% in bi-distilled water. No reactions were observed in the control group treated in the same conditions during the challenge phase. Significant responses in at least 15 % of the test group is considered positive and would be warrant for classification. Therefore the test item tested as described, is considered to be a non-sensitizer.

Mortality:

As there were no deaths during the course of the treatment period no necropsies were performed.

Clinical Signs:

Neither local nor systemic symptoms were observed during the study.

Skin Effect in the Challenge:

Two animals of the test group were observed with defined erythema at the 24-hours reading when treated with the highest non-irritating test article concentration of 50% in bi-distilled water. The same reaction was seen at the 48-hour reading with less severity.

Body weight:

One out of 10 animals of the control group, four out of 20 animals of the test group and 3 out of 4 animals of the irritation screen for challenge lost weight during their acclimatization period. Three out of 4 animals of the irritation screen for induction lost weight during their treatment period. No loss of weight was observed during the treatment phase of the control -, test - and irritation screen for challenge group.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, based on the results obtained from a dermal skin sensitisation test, the test item can be considered to be a non-sensitizer.

Executive summary:

In a dermal sensitization study with the test substance (90% purity) in bi-distilled water (50% w/w), young, male Himalayan guinea pigs (20 animals/test group & 10 animals/control) were tested using the modified Buehler test (OECD 406). Twenty male guinea pigs were treated topically with the test article 50% in bi-distilled water once a week for a three week induction phase. Two weeks after the final induction the animals were challenged with the same test substance used for induction at 50% in bi-distilled water. The animals of the control group were not treated during induction, but were treated once at challenge.  Only two animals of the test group showed a slight confluent erythema at the first (24 h) and a slight, patchy erythema at the second reading (48 h). These findings were considered to be not significant. Therefore, based on the results obtained, the test item can be considered to be a non-sensitizer.