Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Some information in this page has been claimed confidential.

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1998-04-20 to 1998-08-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The tes was conducted before in chemico/ in vitro methods were developed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted by the Council on July 17, 1992 (reported Paris, April 29, 1993)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Juli 30, 1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test substance was tested positive in an LLNA test (Sire, 2006). The test substance is a surfactant with irritating effects. As both properties are described in scientific literature as confounding factors for false positives, the LLNA result is considered inconclusive and an alternative test to the LLNA was thus needed.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: Aqueous solution
Details on test material:
- Physical state: Aqueous solution (clear light yellow liquid)

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: pretest groups: 304-358 g, control and test group: 309-399 g
- Housing: Individually in Makrolon type-4 cages with standard softwood bedding (“Lignocel”, Schill AG, CH-41 32 Muttenz).
- Diet: ad libitum, standard laboratory guinea pig diet Nafag Ecosan 845 25W4, batch nos. 112/97 and 122/97
- Water: ad libitum, Community tap water from Füllinsdorf, once weekly additional supply of ascorbic acid (approx. 1 g/l) via the drinking water was provided.
- Acclimation period: One week for the control and test group under test conditions after health examination. No acclimatization for the animals
of the pretests. Only animals without any visible signs of illness were used.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 38-61 % (mean values: 40-58 %)
- Air changes (per hr): 10-15
- Photoperiod: 12-hour light, 12-hour dark cycle. Music was played during the light period.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
induction:
- intradermal: 5 %
- epidermal: 75 %
challenge: 1 %
Challenge
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
induction:
- intradermal: 5 %
- epidermal: 75 %
challenge: 1 %
No. of animals per dose:
test group: 10
control group: 5
Details on study design:
TEST ARTICLE PREPARATION
The test article and vehicle were placed into a glass beaker on a tared Mettler PM 460 balance and a weight by weight dilution was prepared. Homogeneity of the test article in a suitable vehicle (bi-distilled water) was maintained during treatment using a magnetic stirrer. The preparations were
made immediately prior to each dosing.

RANGE FINDING TESTS:
- Intradermal Injection:
Four intradermal injections (0.1 ml/site) of a 1: 1 (v/v) mixture of Freund’s Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of 5, 3 and 1 % of the test article in bi-distilled water. The resulting dermal reactions were assessed 24 hours later. For intradermal induction application in the main study a 5 % test article concentration was selected.

- Dermal Application:
Four intradermal injections (0.1 ml/site) of a 1: 1 (v/v) mixture of Freund’s Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the 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 A = 100 %, B = 75 %, C = 50 % and D = 25 % in bi-distilled water and applied to the clipped and shaved flanks. The volume of test article applied was approximately 0.2 ml. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. 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, CH-4123 Allschwil) to clean the application site, so that possible erythema reactions were clearly visible at that time.
The depilatory cream 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.

After the above epidermal pretest the highest non-irritating concentration could not be determined. Therefore, a second epidermal pretest was performed in the same manner using two additional guinea pigs and the concentrations of A = 15 %, B = 10 %, C = 5 % and D = 1 % in bi-distilled water.

The concentration selected for the induction period and challenge procedure was 75 % and 1 %, respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
- First stage (intradermal injections): 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.

-- Second stage (epidermal applications): One week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of filter paper was saturated with the test article (75 % in bi-distilled water) and placed between the injection sites of the test animals. The volume of test article applied was approximately 0.3 ml. 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 epidermal application procedure described ensured intensive contact of the test article.

The guinea pigs of the control group were treated as described above with bi-distilled water 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
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 1 % (left flank) and the vehicle only (bi-distilled water applied to the right flank) using the same method as for the epidermal application. The volume of test article applied was approximately 0.2 ml. The dressings were left in place for 24 hours.

Approximately 21 hours after removal of the dressing the test sites treated with the test article were depilated as described in the epidermal pretest.

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. The readings were made under artificial fluorescent light (daylight spectrum).
Challenge controls:
Not applicable
Positive control substance(s):
yes
Remarks:
2-MERCAPTOBENZOTHIAZOLE tested periodically (RCC project 902068) from 27-OCT-1997 to 04-DEC-1997

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
1 % (solid content approx. 0.394%)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1 % (solid content approx. 0.394%). No with. + reactions: 0.0. Total no. in groups: 10.0.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
1 % (solid content approx. 0.394%)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 1 % (solid content approx. 0.394%). No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
1 % (solid content approx. 0.394%)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1 % (solid content approx. 0.394%). No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
1 % (solid content approx. 0.394%)
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 1 % (solid content approx. 0.394%). No with. + reactions: 0.0. Total no. in groups: 5.0.
Group:
positive control
Remarks on result:
not measured/tested

Any other information on results incl. tables

RESULTS:

SKIN EFFECTS AFTER INTRADERMAL INDUCTION -PERFORMED ON TEST DAY 1

- The expected and common findings were observed in the control and test group after intradermal application of FCA in physiological saline and with/without the test article; the intradermal applications were observed with erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

SKIN EFFECTS AFTER EPIDERMAL INDUCTION - PERFORMED ON TEST DAY 8

-CONTROL GROUP: No erythematous or oedematous reaction was observed in the animals treated with bi-distilled water only.

-TEST GROUP: Slight to well-defined erythematous reactions were observed in all test animals treated with the test article at 75 % in bi-distilled water.

SKIN EFFECTS AFTER THE CHALLENGE - PERFORMED ON TEST DAY 22

-CONTROL AND TEST GROUP: No skin reactions were observed in the animals either when treated with bi-distilled water only or when treated with the test article at 1 % in bi-distilled water.

VIABILITY / MORTALITY / MACROSCOPIC FINDINGS

- As there were no deaths during the course of the treatment period no necropsies were performed.

CLINICAL SIGNS, SYSTEMIC

- No symptoms of systemic toxicity were observed in the animals.

BODY WEIGHTS

- The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this adjuvant type guinea pig test method for skin sensitisation conducted according to OECD 406 and GLP, positive reactions in 0 of the 20 animals (0%) were observed during challenge. Therefore, based on the results of this study the substance shall not be classified as a skin sensitiser in accordance with the CLP Regulation.