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Toxicological information

Skin sensitisation

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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:
21 Sep - 15 Oct 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
Adopted in 1992
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The present in vivo study on guinea pigs was performed in 1993 in accordance with the regulatory requirements at that time. The LLNA was not established yet.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
448-100-7
EC Name:
-
Cas Number:
70441-63-3
Molecular formula:
C9H12FN
IUPAC Name:
4-fluoro-N-(propan-2-yl)aniline
Test material form:
liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Hsd/Win:DH (formerly known as Bor:DHPW)
- Source: Harlan Winkelmann GmbH, Borchen (district Paderborn), Germany
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals: SPF-bred
- Age at study initiation: Animals were assumed to be 4 - 7 weeks old, based on body weight data.
- Mean body weight at study initiation: 340 g; range = 284 - 377 g
- Housing: Animals were conventionally housed in Makrolon cages type IV on low-dust wooden granulate (Source: Ssniff Spezialdiäten GmbH, Soest/Westfalen, Germany). Animals were group housed (5 animals/cage during acclimation, 2 - 3 animals/cage during experimental phase).
- Diet: Altromin 3020 - diet for keeping of guinea pigs, Altromin GmbH, Lage, Germany; ad libitum
- Water: tap water of drinking water quality; ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: Animals were examined and described as free of any symptoms.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 40 - 70
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 21 Sep 1993 To: 15 Oct 1993

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Remarks:
test substance was emulsified (w/v) in saline with Cremophor EL (2 % v/v)
Concentration / amount:
intradermal induction: concentration = 1 % (= 4 mg test substance/animal); intradermal injection volume: 0.1 mL
topical induction: concentration: 100 %; epicutaneous treatment with 0.5 mL test substance
Day(s)/duration:
One week after the intradermal injections the topical induction was initiated. Subsquently to the 48 hours epicutaneous exposure period, the test substance was washed off with sterile physiological saline.
Adequacy of induction:
other: Test substance is classified for skin irritation Cat. 2. Signs of skin irritation were not reported/specified for the induction phase. However, it is reported that the day before topical induction, the clipped skin was treated with 10 % SDS in petrolatum.
Challenge
Route:
epicutaneous, semiocclusive
Vehicle:
other: unchanged (no vehicle) or physiological saline with Cremophor EL (2 %)
Concentration / amount:
0.5 mL of 100 % unchanged test substance (no vehicle)
0.5 mL of 50 % test substance formulation in sterile, physiological saline with Cremophor EL (2 %)
No. of animals per dose:
Number of animals in test group: 20
Number of animals in control group: 10
Details on study design:
RANGE FINDING TESTS
In a dose range finding test, one animal was subjected to injections of 0.1 mL volume with the following concentrations: 0, 1, 2.5, and 5 %. Each concentration was injected twice. The injection sites were evaluated 24 and 48 hours post injection. 2.5 and 5 % test substance formulation induced a white area encircled with erythema (1 cm). 1 % test substance formulation caused erythema of 0.5 cm size. Based on these observations 1 % was selected as intradermal induction concentration.
Further, four animals were topically treated with 0.5 mL of undiluted test substance. None of the animals was diagnosed with erythema or edema 48 and 72 hours post application. Based on these observations the undiluted test substance was applied for topical induction.
Similarly, a dose range finding study in five animals resulted in no findings 48 and 72 hours after topical application of the undiluted test substance. Therefore, undiluted test substance was used for the challenge dose.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (one intradermal and one epicutaneous)
- Exposure period: intradermal injection (only on the first study day), and 48 h for epicutaneous exposure
- Test groups:
Intradermal (3 pairs of injections):
Injection 1 (cranial): a 1:1 mixture of FCA/physiological saline
Injection 2 (medial): 1 % test substance in physiological saline with 2 % Cremophor EL
Injection 3 (caudal): 1 % test substance in physiological saline with 2 % Cremophor EL in a 1:1 mixture with FCA

Epicutaneous: undiluted test substance

- Control group:
Intradermal (3 pairs of injections):
Injection 1 (cranial): a 1:1 mixture (v/v) FCA/physiological saline
Injection 2 (medial): physiological saline with 2 % Cremophor EL
Injection 3 (caudal): physiological saline with 2 % Cremophor EL in a 1:1 mixture with FCA

Epicutaneous: physiological saline with 2 % Cremophor EL

- Site: shoulder region (intradermal and epicutaneous)
- Frequency of applications: one intradermal induction at the beginning of the study; one epicutaneous induction one week after the intradermal induction
- Duration: Study days 0-8
- Concentrations: intradermal 1 %, epicutaneous100 %

B. CHALLENGE EXPOSURE
- No. of exposures: one challenge
- Day of challenge: three weeks after intradermal induction
- Exposure period: 24 h
- Test groups: one patch with 100 % test substance, one patch with 50 % test substance in physiological saline with 2 % Cremophor EL (both on the left side of the animal), one patch with vehicle physiological saline with 2 % Cremophor EL only, and one untreated patch (both on the right side of the animal)
- Control group: one patch with 100 % test substance, one patch with 50 % test substance in physiological saline with 2 % Cremophor EL (both on the left side of the animal), one patch with vehicle physiological saline with 2 % Cremophor EL, and one untreated patch (both on the right side of the animal)
- Site: right flank (vehicle and untreated patch) and left flank (both test substance patches)
- Concentrations: 100 % and 50 %
- Evaluation (hr after challenge): 48 and 72 h
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole

Results and discussion

Positive control results:
The reliability of the guinea pig maximisation test was tested in the laboratory with the positive control substance 2-mercaptobenzothiazole, formulated in physiological saline, once per year.
In May 1992, male guinea pigs were subjected to the guinea pig maximisation test with 2-mercaptobenzothiazole. Intradermal induction was performed with a 2.5 %, topical induction with a 40 % positive control substance formulation. After the first provocation with 40 % or 12 % substance formulation, 80 % or 55 % of the test animals reacted with skin lesions, respectively. At the second provocation with 3 % and 1 % substance formulation, 15 % of the test animals showed skin findings each. In the control groups skin redness occurred after no provocation.
In July 1993, the experiment was repeated with 2-mercaptobenzothiazole and the same induction concentrations. As a result, 80 % and 65 % of the test animals reacted to the first provocation with 40 % and 25 % substance formulation and after the second provocation with 12 % and 6 % substance formulation 80 % and 75 % of the test animals, respectively. In the control group, no reaction (no skin redness) was observed.
Sensitivity and reliability of the maximization test methodology are thus confirmed by both studies.
In the results table under "Results and discussion" only the values of the positive control study performed in July 1993 are included.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 %
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
erythema
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 %
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
erythema
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
100 %
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
erythema
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
control patches
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
control patches
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
control patches
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
control patches
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
40%
No. with + reactions:
16
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
25%
No. with + reactions:
13
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
40%
No. with + reactions:
15
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
25%
No. with + reactions:
11
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Remarks:
75% of tested animals showed a positive reaction
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
positive control
Dose level:
12%
No. with + reactions:
14
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
positive control
Dose level:
6%
No. with + reactions:
14
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
72
Group:
positive control
Dose level:
12%
No. with + reactions:
14
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
72
Group:
positive control
Dose level:
6%
No. with + reactions:
14
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

There were no mortalities during the study period. All animals tolerated the treatment without any clinical signs of toxicity. There was no treatment-related effect on body weights.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Following intradermal and topical induction with the test substance, challenge of the treated animals resulted in 50 % positive reactions when applying the test substance at 100 %, and 15 % positive reactions at a test substance concentration of 50 %; controls were negative. Positive control data prove the validity of the method and suitability of the test system. Based on these results, the test substance is considered a skin sensitizer and warrants classification and labelling as Cat. 1, H 317, based on GHS criteria.