Phenothiazine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-03-10 ro 2004-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP study according to OECD TG 406

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Remarks:

GLP compliance statement included in full study report.

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An existing test from 2004 was available.

Test material

Test material information

Constituent 1

Reference substance name:

Phenothiazine

EC Number:

202-196-5

EC Name:

Phenothiazine

Cas Number:

92-84-2

Molecular formula:

C12H9NS

IUPAC Name:

10H-phenothiazine

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST SYSTEM:
- Origin (supplier) of animals: Charles River Deutschland GmbH, Stolzenseeweg 32 - 36, 88353 Kißlegg, Germany
- Animal identification: fur marking with KMnO4 and cage numbering
- Body weight of the main test group at start of study:
mean = 304 g (= 100 %)
min = 273 g (-10.2 %)
max = 334 g (+9.9 %)
n = 15
- Age: Not specified.
- Randomization procedure: Computer generated algorithm (archived with raw data), Randomization schemes 2004.0115.

HOUSING AND CARE OF ANIMALS:
- Animal maintenance: in transparent macrolon cages (type IV) on soft wood granulate in air-conditiones room, 5 animals per cage
- Room temperature: 22 +/- 3 °C (except short lasting deviations due to disturbances of air condition)
- Relative humidity: 50 +/- 20 % (except short lasting deviations due to disturbances of air condition)
- Lighting times: 12 hours light / dark cycle
- Acclimatization: at least five days
- Food: ssniff Ms-H (V 2233), ad libitum
- Water: tap water in plastic bottles, ad libitum

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: sesame oil

Induction 2

Concentration / amount:

INTRADERMAL INDUCTION:
1) 0 % of test substance in 50 % Freund's Adjuvants
2) 5 % of test substance in sesame oil
3) 5 % of test substance in 50 % Freund's Adjuvants

DERMAL INDUCTION:
25 % of the test substance in sesame oil

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: sesame oil

Challenge 2

Concentration / amount:

INTRADERMAL INDUCTION:
1) 0 % of test substance in 50 % Freund's Adjuvants
2) 5 % of test substance in sesame oil
3) 5 % of test substance in 50 % Freund's Adjuvants

DERMAL INDUCTION:
25 % of the test substance in sesame oil

No. of animals per dose:

Treatment group: 10
Control group: 5

Details on study design:

RANGE FINDING TESTS:
Determination of the tolerance of the intradermal injections:
- 2 animals
- 3 conc. of test substance in sesame oil: 5.0 %, 1.0 %, 0.2 % administered twice each to each animal by intradermal injections.
- 24, 48, 72 and 96 hours after administration the injection sites were examined for local tolerance.

Determination of the primary non-irritant concentration:
- 3 animals
- each animal received 4 intradermal injections of a 50% Freund's Complete Adjuvant emulsion
- administration of test substance by cellulose patch (occlusive) in sesame oil for 24 hours
- Dosage:
Animal No Left flank Right flank
1 25% 5%
2 25% 1%
3 5% 1%
- Examination of treated skin areas for erythema and edema according to Draize 24 hours after removal of pathces

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (one intradermal injection (day 1 and one dermal treatment by occlusive patch (day 8))
- Exposure period: Intradermal injection on day 1, next administration (dermal, occlusive) on day 8 for 48 hours.
- Test groups: One (10 animals)
- Control group: One (5 animals)
- Site: Intradermal: dorsal site; Dermal: site of intradermal injection.
- Number/Frequency of applications: Intradermal: two injection per animal; dermal: one application
- Duration: Dermal: 48 hours
- Concentrations:
Intradermal induction - Treatment group:
Site Appl. Vol [mL] Conc. [%] Preparation
1 2 x 0.1 -- 50% Freund's Adjuvants
2 2 x 0.1 5 Substance in sesame oil
3 2 x 0.1 5 Substance in 50% Freund's Adjuvants

Intradermal induction - Control group:
Site Appl. Vol [mL] Preparation
1 2 x 0.1 50% Freund's Adjuvants
2 2 x 0.1 Sesame oil
3 2 x 0.1 Equal volume of sesame oil and 50% Freund's Adjuvants

B. CHALLENGE EXPOSURE
- No. of exposures: One
- Day(s) of challenge: Day 22
- Exposure period: 24 hours
- Test groups: One
- Control group: One
- Site: Left flank
- Concentrations: 25 % Phenothiazin D Prills in sesame oil
- Evaluation (hr after challenge): Examination of the skin approx. 24 as well as 48 hours after removal of the patches.


OTHER:
- Day 29: Body weight of the test animals was determined.

Challenge controls:

Separate study: Positive control assay, PT03-0397, 23-Feb-2004

Positive control substance(s):

yes

Remarks:

Alpha-hexyl cinnamic aldehyde

Results and discussion

Positive control results:

After the challenge treatment ten animals of the treatment group (100%) showed a positive reaction during the observation period (Positive control assay, PT03-0397, 23-Feb-2004).

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

None.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

25%

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

none

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

25%

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

none

Any other information on results incl. tables

Results: Determination of the tolerance of the intradermal injections

- Intradermal injections with the 5.0% preparation caused slight erythema and edema.

- Based on this preliminary test, a 5% preparation was selected for the intradermal injections in the main test.

Results: Determination of the primary non-irritating concentration

- No signs of irritation occurred after administration of the different test concentrations.

- Based on these results, a concentration of 25% Phenothiazin D Prills in sesame oil was chosen for the challenge at day 22.

Intradermal induction treatment

The administration sited treated without test substance showed severe erythema and edema. Intradermal injections with Freund's Adjuvant (with test substance) caused severe erythema and edema as well as encrustations. The administration sites treated with the test substance in sesame oil showed slight erythema and edema. Intradermal injections of the vehicle alone exhibited no signs of irritation.

Due to these strong irritation reactions of the skin, 10% sodium dodecylsulfate was not administered at day 7.

Dermal induction treatment

After removal of the patches at day 10, severe erythema and edema, indurated, scabbed and encrusted skin as well as necrosis were observed at the site previously treated with Freund's Adjuvant. The administration sites treated with the test substance or the vehicle alone showed no signs of irritation.

Challenge treatment – control and treatment group

Table 1: individual values (1st reading, 24 hours)
Control animal No:	1	2	3	4	5
Value	0	0	0	0	0
Treated animal No	6	7	8	9	10	11	12	13	14	15
Value	0	0	0	0	0	0	0	0	0	0

 

Table 1: individual values (2nd reading, approx. 48 hours)
Control animal No:	1	2	3	4	5
Value	0	0	0	0	0
Dry and rough			x
Treated animal No	6	7	8	9	10	11	12	13	14	15
Value	1	0	0	0	1	1	0	0	1	0
Dry and rough	x			x	x	x	x	x	x	x
Chapped					x	x	x		x

 

After the challenge treatment 4 animals of the treatment group (40%) showed a positive reaction during the observation period. Scaling of dermal reactions according to the score of DRAIZE, with 1 representing very slight findings (barely perceptible).

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

Under the conditions of the present study, 4 out of ten animals of the tratment group showed a weak positive response after the challenge procedure (48 hours).
Based on the results of this study Phenothiazin D Prills may cause sensitization by skin contact according to the classification criteria of Directive 2001/59/EC, and ECETOC-Monograph No. 29 (Brussels, Sept. 2000).

Executive summary:

Summary

Skin sensitization of Phenothiazin D Prills was performed in female guinea pigs according to the method of Magnusson & Kligman.

Intradermal induction was performed using 5% Phenothiazin D Prills in sesame oil. Dermal induction and challenge treatment were carried out with 25% Phenothiazin D Prills in sesame oil.

The validity of the test system is confirmed by the periodically conducted positive control test using Alpha-hexylcinnamaledhyde for the maximization test (report number PT03 -0397, dated 23 -Feb-2004; Aventis Pharma Deutschland GmbH, ProTox).

Based on the results of this study Phenothiazin D Prills may cause sensitization by skin contact according to the classification criteria of Directive 2001/59/EC, and ECETOC-Monograph No. 29 (Brussels, Sept. 2000).