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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:
December 29, 1993 - February 02, 1994
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1993
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1992
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study (different from LLNA test) is available.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CHARLES RIVER Wiga GmbH, Sandhofer Weg 7, D-97633 Sulfeld.
- Age at beginning of acclimatization period: 6-7 weeks.
- Weight at beginning of acclimatization period: control and test group at 321 - 371 g; pretest at 326 - 390 g.
- Housing: individually in Makrolon type-3 cages (22x37x15 cm) with standard softwood bedding ("lignocel", Schill AG, CH-4132 Muttenz).
- Diet: pelleted standard Kliba 341, Batch 83/94 rabbit maintenance diet ("Kllba", Klingentalmueble AG, CH-4303 Kaiseraugst), ad libitum.
- Water: community tap water from Fullinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/L) via the drinking water. A bacteriological, chimical and contaminant analyses were performed.
- Acclimation period: one week for the control and test group under test conditions after health examination. One day for the animals of the intradermal pretest and 5 days for the animals of the epidermal pretest. Only animals without any visual signs of illness were used for the study.
- Identification: by unique cage number and corresponding ear tags.
- Randomization: randomly selected at time of delivery.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 40-60 %; during the cleaning process (approx. 30 min) could have been above 70 %
- Air changes: 10-15 ACH
- Photoperiod: 12 hours fluorescent light (approx. 100 lux) / 12 hours dark; music during the light period.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Two patches (2 x 2 cm) of filter paper saturated with the highest non-irritating concentration of 25 % (left flank).
Challenge
Route:
epicutaneous, semiocclusive
Vehicle:
water
Concentration / amount:
Two patches (2 x 2 cm) of filter paper saturated with the highest non-irritating concentration of 25 % (left flank).
No. of animals per dose:
MAIN TEST: 30 males (10 control group + 20 test group)
PRE-TEST: 6 males (2 intracutaneous pretest + 4 epicutaneous pretest)
Details on study design:
MAIN STUDY
Intradermal injections / performed on test day 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. 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 as follows:
Test group:
1) 1:1 (v/v) mixture of Freund’s Complete Adjuvant and physiological saline
2) The test article, diluted to 5% with bi-distilled water
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) Bi-distilled water
3) 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline

Epidermal applications / 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 dipped, shaved freeof hair and the test area was pre-treated with 10 % Sodium-Lauryl-Sulphate (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.
On test day 8 a 2 X 4 cm patch of filter paper was saturated with the test article (25% in bi-distilled water) and placed over the injection sites of the test animals. The patch was covered with aluminium 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 approximately 48 hours. The epidermal application procedure described ensured intensive contact of the test article.
The guinea-pigs of the control group were treated as described above with bidistilled water.
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.

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.
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. Two patches (2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 25 % (left flank) and the vehicle only (bi-distilled water applied to the right flank) using the same method as for the epidermal application. The dressing were left in place for 24 hours. 21 hours after removing of the dressing the test sites were depilated with an approved depilatory cream. The cream was placed on the patch sites for 3-5 minutes and then washed off with a stream of warm running water. When the application sites were clean and any stains from the test article removed theanimals were dried with a disposable paper towel and returned to their cages.
Approximately 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.
Challenge controls:
The most recent test was run from November 15, 1993 to December 28, 1993
Positive control substance(s):
yes
Remarks:
4-Aminobenzoic acid ethyl ester in a current reference study

Results and discussion

Positive control results:
The intradermal induction was performed with a dilutlon at 1 % in mineral oil. For the induction period and challenge procedure a 25 % dilution of 4-Aminobenzoic acid ethyl ester in mineral Oil was used. This concentratlon was found in a pretest to be the highest primary not-irritant concentration. The test article at 10 and 5 % in mineral oil was additionally applled In the second challenge to compare with the literature.
From the results described above moderate allergenic potency (grade 3) of the test article 4-Aminobenzoic acid ethyl ester was concluded. The results were interpreted according to the rating of Magnusson and Kligman (1969).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 % test article in bi-distilled water
No. with + reactions:
14
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25 % test article in bi-distilled water
No. with + reactions:
12
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25 % test article in bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 % test article in bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
4-aminobenzoic acid ethyl ester, 25 % in mineral oil
No. with + reactions:
10
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
4-aminobenzoic acid ethyl ester, 25 % in mineral oil
No. with + reactions:
9
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
positive control
Dose level:
4-aminobenzoic acid ethyl ester, 10 % in mineral oil
No. with + reactions:
8
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
positive control
Dose level:
4-aminobenzoic acid ethyl ester, 10 % in mineral oil
No. with + reactions:
7
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
positive control
Dose level:
4-aminobenzoic acid ethyl ester, 5 % in mineral oil
No. with + reactions:
6
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
positive control
Dose level:
4-aminobenzoic acid ethyl ester, 5 % in mineral oil
No. with + reactions:
6
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

No. erythema detected at first challenge procedure (out of 20 animals)

Control: 0/10

After 24 hours at 25 %: 10/20 (left flank)

After 48 hours at 25 %: 9/20 (left flank)

No. erythema detected at second challenge procedure (out of 20 animals)

Control: 0/10

After 24 hours at 10 %: 8/20 (right cranial flank)

After 24 hours at 5 %: 6/20 (right caudal flank)

After 48 hours at 10 %: 7/20 (right cranial flank)

After 48 hours at 5 %: 6/20 (right caudal flank)

Test group skin response after the challenge procedure. Treated wth test article at 25 % in bi-distilled water (left flank)

Animal no. Sex Erythema and Oedema readings 
After 24 hours After 48 hours
E Oe E Oe
411 M 2 1 2 1
412 M 0 0 0 0
413 M 0 0 0 0
414 M 2 1 2 0
415 M 1 0 1 0
416 M 1 0 1 0
417 M 0 0 1 0
418 M 1 0 0 0
419 M 1 0 1 0
420 M 2 0 1 0
421 M 2 1 2 1
422 M 1 0 0 0
423 M 1 0 0 0
424 M 1 0 1 0
425 M 1 0 1 0
426 M 0 0 0 0
427 M 1 0 1 0
428 M 0 0 0 0
429 M 0 0 0 0
430 M 2 1 2 1
Total positive 14/20 4/20 12/20 3/20

Applicant's summary and conclusion

Interpretation of results:
other: sub-category 1B according to the CLP Regulation (EC 1272/2008)
Conclusions:
The substance was found to have a skin sensitising potential.
Executive summary:

The test was performed according to OECD guideline 406 (1993). To assess the allergenic potential of test item in albino guinea pigs the Maximization-Test of B. Magnusson and A.M. Kligman (1969) was used. Ten males were used as control group and 20 males were used as test group.

In the study it was found that 70 and 60% of the animals gave a positive response at the 24 and 48 hour time points respectively following challenge with the highest non-irritating concentration of 25 % test item.