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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-11-15 until 1993-12-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted according to Guideline and under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
Buehler test

Test material

Constituent 1
Chemical structure
Reference substance name:
3-chloro-o-toluidine
EC Number:
201-756-6
EC Name:
3-chloro-o-toluidine
Cas Number:
87-60-5
Molecular formula:
C7H8ClN
IUPAC Name:
3-chloro-2-methylaniline
Details on test material:
- name of the test substance: 3-Chlor-2-methylanilin
- Product number / Code: HOE CG 0221 OD ZD99 0001
- Synonyma: 6-Chlor-2-toluidin
- Chemical name: Benzolamin, 3-Chlor-2-methyl-
- Solubility: > 10 g/L soluble in DMSO, ethanol and acetone
- Purity: 98.8 %
- Batch and production date: strassentankzug / 2 (Riedel), March 1990
- Certificate of analysis: No. 04877 from January 10th, 1991
- Storage conditions: darkness at room temperature in a fume cupboard
- Storage stability: stable at least until December 1995.
- Stability and homogeneity in the vehicle: is guaranteed for 4 hours

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Pirbright-White, Hoe: DHPK (SPFlac)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Body weight at study initiation: 282-331 g
- Housing: in groups of 5 animals
- Diet: Altromin 3112 for guinea pigs and rabbits ad libitum
- Water: tap water ad libitum
- Acclimation period: at least one day (5 days not necessary; breeding at identical conditions)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: undiluted (dermal induction and first challenge) or mixed with petrolatum, DAB (second challenge treatment).
Concentration / amount:
- Determination of the primary non-irritant concentration: 100 % 3-chloro-2-methylaniline (3C2M); 25 % 3C2N in petrolatum; 5 % 3C2M in semi-liquid paraffin; 1 % 3C2M in semi-liquid paraffin.
- Dermal inductions and first challenge: 100 % 3-chloro-2-methylaniline
- Second challenge: 50 % 3-chloro-2-methylaniline was chosen due to irritations during dermal induction after 3rd treatment.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: undiluted (dermal induction and first challenge) or mixed with petrolatum, DAB (second challenge treatment).
Concentration / amount:
- Determination of the primary non-irritant concentration: 100 % 3-chloro-2-methylaniline (3C2M); 25 % 3C2N in petrolatum; 5 % 3C2M in semi-liquid paraffin; 1 % 3C2M in semi-liquid paraffin.
- Dermal inductions and first challenge: 100 % 3-chloro-2-methylaniline
- Second challenge: 50 % 3-chloro-2-methylaniline was chosen due to irritations during dermal induction after 3rd treatment.
No. of animals per dose:
- Determination of primary non-irritant concentration: 6
- Control group: 10
- Treated group: 20
Details on study design:
- Determination of primary non-irritant concentration:
The hair on the flank of the animals was removed mechanically. 0.5 mL or g of the test substance preparation was applied to a 2 x 2 cm cellulose patch, which was fixed to the left flank (100% test substance on the right flank) and covered occlusively (with film and bandage) for 6 hours. The treated skin area was examined 24 hours after removal of the patches for erythema and oedema according to the technique of DRAIZE.

- Main test for sensitizing properties:
Day 1 to 15
During this test phase, the following procedures were carried out once a week. For the dermal treatments, 0.5 mL of 100 % 3-Chlor-2-methylanilin was applied evenly over a 2 x 2 cm cellulose patch, which was fixed to the front part of the left flank of the 20 animals of the treatment group and then covered with an occlusive polyethylene film and a bandage (Fixomull). The 10 control animals were treated analogously with 0.5 mL semi-liquid paraffin. After an exposure period of 6 hours the occlusive bandage was removed and the flank skin washed. Clinical signs and irritant effects emerging during the sensitization phase were recorded.

Days 16 to 28
No further animal treatment. Animals under observation (weekends excepted).

Day 29
Challenge treatment. This took place under identical conditions for control and treated animals alike. The hair on the previously untreated right flank was removed mechanically. Challenge treatment was performed with 100 % 3-Chlor-2-methylanilin. 0.5 mL of the test substance was applied to a cellulose patch, which was placed on the skin and covered with an occlusive bandage. Exposure was for 6 hours. After removal of the occlusive bandage, any remnants of the substance were carefully washed off with warm water.

Day 30 and 31
Macroscopic examination of the skin.

Day 36
Second challenge treatment. Treatment was performed in the same way as the first challenge treatment. 50% 3-Chlor-2-methylanilin in petrolatum was chosen because of irritations during dermal induction after 3rd treatment.

Day 37 and 38
Macroscopic examination of the skin.

Day 38
Terminal body weight determination.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100% 3-chloro-2-methylanilin
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Erythema score 2 in both animals.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100% 3-chloro-2-methylanilin. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: Erythema score 2 in both animals..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100% 3-chloro-2-methylanilin
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100% 3-chloro-2-methylanilin. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100% 3-chloro-2-methylanilin
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: 100% 3-chloro-2-methylanilin. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%3-chloro-2-methylanilin
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100%3-chloro-2-methylanilin. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
50% 3-chloro-2-methylanilin
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
Erythema score 2 in all positive animals.
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 50% 3-chloro-2-methylanilin. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: Erythema score 2 in all positive animals..
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
50% 3-chloro-2-methylanilin
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
Erythema score 2 in 2 animals and 1 in the third animal.
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 50% 3-chloro-2-methylanilin. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: Erythema score 2 in 2 animals and 1 in the third animal..
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
50% 3-chloro-2-methylanilin
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 50% 3-chloro-2-methylanilin. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
50% 3-chloro-2-methylanilin
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 50% 3-chloro-2-methylanilin. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

1st and 2nd dermal induction:

- Erythema in control animals: 0/10

- Edema in control animals: 0/10

- Erythema in test animals: 0/20

- Edema in test animals: 0/20

3rd dermal induction:

- Ertythema in control animals: 0/10

- Edema in control animals: 0/10

- Erythema in test animals: 6/20

- Edema in test animals: 1/20

There were no recognizable differences in body weight gains between the control and treated animals. No clinical signs of intoxication were observed at any time during the study. Dry-rough skin was observed in two animals of the treatment group after the 2nd application. Well-defined erythema, very slight oedema, fine scales, encrustations as well as dry rough skin occurred sporadically in the treatment group during the sensitization phase after the 3rd application. The animals of the control group showed no signs of irritation.

After first challenge treatment two animals of the treatment group showed well-defined erythema 24 hours after removal of the patches.

Second challenge treatment exhibited very slight to well-defined erythema, dry rough skin and fine scales in three animals of the treatment group 24 and 48 hours after removal of the patches. The animals of the control group showed no signs of irritation after the first and second challenge treatment.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of this study, the test substance 3-chloro-2-methylaniline needs to be classified Skin Sensiting R43 and Catagory 1B according to Directive EEC/67/548 and regulation (EU) No. 1272/2008, respectively.