5-chloro-2-(2,4-dichlorophenoxy)aniline

Skin sensitisation

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:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

5-chloro-2-(2,4-dichlorophenoxy)aniline

EC Number:

260-480-4

EC Name:

5-chloro-2-(2,4-dichlorophenoxy)aniline

Cas Number:

56966-52-0

Molecular formula:

C12H8Cl3NO

IUPAC Name:

5-chloro-2-(2,4-dichlorophenoxy)aniline

Details on test material:

- Name of test material (as cited in study report): FAT 80'023/G (TADE)
- Physical state: Grey-green solid
- Analytical purity: > 98%
- Lot/batch No.: ICMD Juli 95
- Expiration date of the lot/batch: 31-AUG-2000
- Stability under test conditions: Stable in polyethylene glycol (PEG 400) for at least 48 hours; the stability is unknown in a 1:1 (v/v) mixture of FCA/physiological saline and therefore is excluded from the statement of compliance.
- Storage condition of test material: In the original container at room temperature (approx. 20°C), away from direct sunlight.

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Ibm: GOHI; (synonym: Himalayan spotted)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Fuellinsdorf/Switzerland
- Age at study initiation: 5 - 7 weeks
- Range of body weight at study initiation: 363 - 595 g; Mean body weight at day 1 of treatment: control group: 444 ± 34.0 g; test group: 416 ± 27.5 g.
- Housing: Individually in Makrolon type-3 cages
- Diet (e.g. ad libitum): ad libitum
- Water: tap water, ad libitum
- Acclimation period: One week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22 °C
- Humidity (%): 46-56%.
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light, 12-hour dark cycle.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

polyethylene glycol

Induction 2

Concentration / amount:

For intradermal induction application, a 5% test article dilution was selected.
The concentration selected for the induction period and challenge procedure was 75%.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

polyethylene glycol

Challenge 2

Concentration / amount:

For intradermal induction application, a 5% test article dilution was selected.
The concentration selected for the induction period and challenge procedure was 75%.

No. of animals per dose:

30 females, nulliparous and non-pregnant; control: 10 females; test group: 20 animals.

Details on study design:

RANGE FINDING TESTS:
The objective was to identify a maximally tolerated concentration of the test article suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application.

A) INTRADERMAL INJECTIONS:
Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1% of the test article in PEG 400.
The resulting dermal reactions were assessed 24 hours later.
For intradermal induction application a 5% test article dilution was selected.

B) EPIDERMAL APPLICATIONS:
Both flanks of each of 4 guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper ( 2 x 2 cm) were saturated with the test article at 75% (this concentration used was found to be the most qualified to assure an optimum technical application procedure), 50%, 25% and 15% in PEG 400 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 impervious adhesive tape. The dressings were removed after an exposure period of 24 hours.
Approximately 21 hours after removal of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, Allschwil, Switzerland) to facilitate the evaluation of a possible erythematous reaction.
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 then dried with a disposable towel, and returned to their cages.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize.
The concentration selected for the induction period and challenge procedure was 75%.

MAIN STUDY
A. INDUCTION EXPOSURE
a) Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region.
- No. of exposures: 1
- Exposure period: 7 days
- Test group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, diluted to 5% with PEG 400.
3) The test article 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) PEG 400.
3) 1:1 (w/w) mixture of PEG 400 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

- Site: dorsal
- Frequency of applications: once

b) Epidermal induction performed on test day 8:
On test day 7 and approximately 24 hours prior to the epidermal application the scapular area (approximately 6 x 8 cm) was clipped, shaved free of hair and the test area was prepared with 10% Sodium-Lauryl-Sulfate (SLS) in paraffinum perliquidum as no primary irritation had been observed in the pretest. The SLS was massaged into the skin with a glass rod without bandaging. This 10% concentration of SLS enhances sensitization by provoking a mild inflammatory reaction.
One week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair. A 2 x 4 cm patch of filter paper was saturated with the test article (75% in PEG 400) and placed over the injection sites of the test animals. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 48 hours.
The guinea-pigs of the control group were treated as described above with PEG 400 only. Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.

B. CHALLENGE EXPOSURE
CHALLENGE / performed on test day 22
The test and control guinea-pigs were challenged two weeks after the epidermal induction application. The test and control guinea-pigs were treated in the same way.
- No. of exposures: once
- Test groups: treatment with 75% test compound
- Control group: vehicle only (PEG 400)
- Site: Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea-pig just prior to the application.
- Concentrations: Two patches ( 2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 75% (left flank) and the vehicle only (PEG 400 applied to the right flank) using the same method as for the epidermal application.
- Exposure period: The dressings were left in place for 24 hours.
- Evaluation (hr after challenge): 24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize (Draize J.H., Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, Association of Food and Drug Officials of the United States, Austin, Texas, 1959)

OTHER:
- TEST ARTICLE PREPARATION
The test article and vehicle were placed into a glass beaker on a tared Mettler PM 480 balance and weight/weight dilutions were prepared. Homogeneity of the test article in a suitable vehicle* was maintained during treatment using a magnetic stirrer. The preparations were made immediately prior to each dosing.

* PEG 400 was used for the intradermal and epidermal pretests. It was also used for the intradermal and epidermal induction and the challenge in the main study. The 1:1 mixture (v/v) of Freund's Complete Adjuvant:physiological saline was used as a vehicle for the intradermal induction in the main study.

Positive control substance(s):

not required

Remarks:

separate studies were performed with 4-aminobenzoic acid ethyl ester and 2-Mercaptobenzothiazol

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75 %

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

No symptoms of systemic toxicity were observed in the animals.

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75 %. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: No symptoms of systemic toxicity were observed in the animals..

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Based on the study results the substance is considered to be not sensitizing.

Executive summary:

In this study 5% of the animals of the test group were observed with positive skin reactions at the 24-hour reading after treatment with a non-irritant test substance concentration of 75% in PEG 400. No skin reactions were observed in the control group.

Therefore, the test article FAT 80'023/G (TADE) at concentration of 75% in PEG 400 is considered to be a non-sensitizer when described under the test conditions.