4-chloro-2,5-dimethoxyaniline

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-11-15 to 2001-02-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP-study according to OECD TG.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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

GLP compliance:

yes

Remarks:

GLP compliance statement included in full study report.

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The original Test Guideline (TG) for the determination of skin sensitization in the mouse, the Local Lymph Node Assay (LLNA; TG 429)
was adopted in 2002. This study was performed earlier - in 2000.

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-2,5-dimethoxyaniline

EC Number:

228-782-0

EC Name:

4-chloro-2,5-dimethoxyaniline

Cas Number:

6358-64-1

Molecular formula:

C8H10ClNO2

IUPAC Name:

4-chloro-2,5-dimethoxyaniline

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Pirbright-White

Sex:

female

Details on test animals and environmental conditions:

- Source: Harlan Winkelmann, D-33178 Borchen, SPF breeding colony
- Strain: HsdPoc:DH
- Body weight of the main test group at start of study: Mean = 380g (min: 360g, max: 432g)
- Randomization: Randomization scheme 2000.0453
- Animal maintenance: in fully air-conditioned rooms in macrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Room temperature: 20 +/- 3°C
- Rel. humidity: 50 +/- 20 %
- Lighting time: 12 hrs daily
- Acclimatization: at least 7 days
- Diet: ssniff (R) Ms-H (V2233), ad libitum
- Water: tap water in plastic bottles, ad libitum
Animal identification: fur marking with KMnO4 and cage numbering

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: hydroxyethyl-cellulose (Tylose H 4000 G4 PHA)

Induction 2

Concentration / amount:

Intradermal induction treatment: 5% test substance in 0.5% aqueous solution of vehicle and 5% test substance in 50% Freund's Adjuvant
Dermal induction treatment: 25% test substance in 0.5% aqueous solution of vehicle
Dermal challenge treatment: 25% test substance in 0.5% aqueous solution of vehicle

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: hydroxyethyl-cellulose (Tylose H 4000 G4 PHA)

Challenge 2

Concentration / amount:

Intradermal induction treatment: 5% test substance in 0.5% aqueous solution of vehicle and 5% test substance in 50% Freund's Adjuvant
Dermal induction treatment: 25% test substance in 0.5% aqueous solution of vehicle
Dermal challenge treatment: 25% test substance in 0.5% aqueous solution of vehicle

No. of animals per dose:

Determination of the tolerance of the intradermal injections: 2
Determination of the primary non-irritating concentration: 3
Control group: 5
Treatment group: 10

Details on study design:

RANGE FINDING TESTS:
- Determination of the tolerance of the intradermal injections: 2 animals, concentrations tested: 5, 1 and 0.2 % of test substance in 0.5% w/w aqueous solution of vehicle.
- Determination of the primary non-irritating concentration: 3 animals, concentrations tested: 25, 5 and 1 % of test substance in 0.5% w/w aqueous solution of vehicle.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: two intradermal injections on day 1, one dermal on day 8
- Exposure period: dermal: between day 2 and day 7: administration areas were examined for local tolerance. Systemic toxic effects were noted, if apparent
- Test groups: 1
- Control group: 1
- Site: dorsal area, in vicinity of shoulders
- Frequency of applications: 1

B. CHALLENGE EXPOSURE
- Dermal challenge treatment start on day 22
- No. of exposures: 1
- Day(s) of challenge: day 22 - day 25 (cahallenge and post-challenge observation)
- Exposure period: 24 hrs, then occlusive bandagae was removed.
- Test groups: 1
- Control group: 1
- Site: left flank
- Evaluation (hr after challenge): 48 and 72 hrs


OTHER:

Positive control substance(s):

yes

Remarks:

The validity of the test system is confirmed periodically by using alpha-hexyl cinnamic aldehyde (Report No 2000.0738, 2000-10-20, Aventis Pharma Deutschland GmbH, ProTox)

Results and discussion

Positive control results:

The validity of the test system is confirmed periodically by using alpha-hexyl cinnamic aldehyde (Report No 2000.0738, 2000-10-20, Aventis Pharma Deutschland GmbH, ProTox)

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.

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None..

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None..

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

other: control group

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None.

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: control group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None..

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

other: Control group

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None.

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None..

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Under the conditions of the present study, none of ten animals of the treatment group showed a positive skin response after the challenge procedure.
Thus, the percentage of animals reacting positive is below the threshold of 30 %. Based on the results of this study 4-chloro-2,5-dimethoxyaniline showed no evidence for sensitising properties according to the classification criteria of Directive 93/21/EEC.

Executive summary:

Testing for sensitising properties of 4 -chloro-2,5 -dimethoxyaniline was performed in female guinea pigs according to the method of Magnusson & Kligman. Intradermal induction was performed using 5% 4 -chloro-2,5 -dimethoxyaniline in 0.5% w/w aqueous solution of hydroxyethyl-cellulose (Tylose H4000 G4 PHA). Dermal induction and challenge treatment were carried out with 25% 4 -chloro-2,5 -dimethoxyaniline in 0.5% w/w aqueous solution of hydroxyethyl-cellulose (Tylose H4000 G4 PHA).

The validity of the test system is confirmed by periodically conducted positive control test using alpha-hexyl cinnamic aldehyde for the Buehler test (report number 2000.0738, dated October 20, 2000; Aventis Pharma Deutschland GmbH, ProTox). Based on the results of this study 4 -chloro-2,5 -dimethoxyaniline showed no evidence for sensitising properties according to the classification criteria of Directive 93/21/EEC.