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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:
10/01/91 - 24/12/91
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
not specified
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Justification for non-LLNA method is not available within LoA data availability.

Test material

Constituent 1
Chemical structure
Reference substance name:
N-cyclohexyl-N-methylcyclohexylamine
EC Number:
231-453-4
EC Name:
N-cyclohexyl-N-methylcyclohexylamine
Cas Number:
7560-83-0
Molecular formula:
C13H25N
IUPAC Name:
N-cyclohexyl-N-methylcyclohexanamine
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: POLYCAT 12; lot: 70891CJ
- Expiration date of the lot/batch: not indicated
- Purity: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stability in corn oil not indicated by the sponsor; stable under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle:
The choice of corn oil as vehicle in this test was based on the fact that the test article diluted well in corn oil at the concentrations used; stability in corn oil not indicated by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Prior to each treatment, the test substance was weighed into small glass containers, corn oil was added (w/w) and subsequently mixed using a mechanical
stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
mixture in corn oil

OTHER SPECIFICS:
clear colourless to amber liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Remarks:
Albino
Sex:
female
Details on test animals and environmental conditions:
- Source: BRL Ltd., Basel, Switzerland
- Microbiological status of animals, when known: SPF- quality
- Age at study initiation: approx. 9 weeks
- Weight at study initiation: 359 – 462 g
- Housing: group housing of 2 animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system
- Diet (e.g. ad libitum): free access to standard guinea pig diet, including ascorbic acid (1600 mg/kg); LC 23-B, pellet diameter 4mm. (Hope Farms, Woerden, The Netherlands). In addition, hay (Broekman Institute, Someren, The Netherlands) was provided once a week.
- Water (e.g. ad libitum): free access to tap-water, diluted with decalcified water
- Acclimation period: at least 5 days before start of treatment


ENVIRONMENTAL CONDITIONS
Standard Laboratory Conditions:
- Temperature (°C): 21°C
- Humidity (%): 55%
- Air changes (per hr): 15
Fluctuations from the optimal conditions were noted, but were considered not to have affected study integrity
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
corn oil
Concentration / amount:
5% w/w
Day(s)/duration:
7 days
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 ml
Day(s)/duration:
2 days
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
50% w/w
Day(s)/duration:
1 day
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
25% w/w
Day(s)/duration:
1 day
Adequacy of challenge:
highest non-irritant concentration
No.:
#3
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
10% w/w
Day(s)/duration:
1 day
Adequacy of challenge:
highest non-irritant concentration
No.:
#4
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
0%
Day(s)/duration:
1 day
No. of animals per dose:
Preliminary study: 5 females
Experimental group: 20 females
Control group: 10 females
Details on study design:
RANGE FINDING TESTS:
Primary irritation experiment
Intradermal injections:
Four intradermal injections (0.1 ml/site) were made into the clipped shoulder region of one guinea pig at a concentration of 5 % (w/w) of the test substance in corn oil. The resulting dermal reactions were assessed 24 and 48 hours later.
The following parameters were recorded:
Erythema - scored according to the scale (see Any other information ...)
Necrosis - if present yes or no,
Diameter - (mm) of effect (erythema, necrosis or discolouration).

Epidermal applications:
The intradermally injected animal was also treated epidermally at the shaved left flank with 0.5 ml of a concentration of 50 % test substance in corn oil using a Scotchpak-non-woven patch (2.5 x 2.2 cm) mounted on Micropore tape (3M, U.S.A.) and held in place with Coban elastic bandage (3M, U.S.A.). After 24 hours, the dressings and residual test article were removed using a moistened tissue. The treated skin was assessed for erythema and oedema 24 and 48 hours after bandage removal on a numerical basis according to the scale described below.
Four other animals were shaved on the left flank and exposed to 0.05 ml of a 50 %, 25 %, 10 % and 5 % (w/w) test substance concentration in corn oil, occlusively administered by means of Square chambers (v.d. Bend, Brielle, The Netherlands) mounted on Micropore tape and fixed in place by means of Coban elastic bandage. This procedure ensured the intensive contact of the test substance even if it is insoluble in the vehicle used. After 24 hours, the dressings and residual test article were removed using a moistened tissue.
The reaction sites were assessed for erythema and oedema on a numerical basis according to the scale described below, 24 and 48 hours after bandage removal.
Immediately after the 24 hour skin reading the treated areas were shaved.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures:
3x2 of intradermal injections 0.1 ml/site
7 days after the intradermal injections patch of Scotchpak non-woven (2x4 cm) mounted on Micropore tape was applied with 0.5 ml of the undiluted test substance and placed between the injection sites of the test animals. After 48 hours, the dressings and residual test article were removed using a moistened tissue.
Because the test substance was applied undiluted for the epidermal induction, a dry patch was administered to the test area of the control animals.
- Exposure period: day 1, day 8
- Test groups: 20
- Control group: 10
- Site: the dorsal skin from the scapular region 2x4 cm area (clipped)
- Frequency of applications:
- Duration: 7 days + 2 days
- Concentrations:
A. 5% w/w with corn oil
B. Freunds' Complete Adjuvant (FCA; Difco, Detroit, U.S.A.), 50:50 with distilled water for injection (pyrogen free)
C. 10% w/w test substance as mixture with FCA 50:50

Reaction sites were assessed for erythema and oedema immediately after removal of the dressings, using the numerical grading system (see Any other information ...)

B. CHALLENGE EXPOSURE
- No. of exposures: 4
- Day(s) of challenge: The dressings and residual test substance were removed after approximately 24 hours, using a moistened tissue.
- Exposure period: 2 weeks after the epidermal induction application
- Test groups: 20 animals
- Control group: 10 animals
- Site: a 5 x 5 cm area on the left flank of each guinea pig (clipped and shaved)
- Concentrations: 0.05 ml of each of the following 3 test substance concentrations and the vehicle were applied using Square chambers attached to Micropore tape
a = 50 % (w/w) in corn oil
b = 25 % (w/w) in corn oil
c = 10 % (w/w) in corn oil
d = in corn oil
- Evaluation (hr after challenge):
The sites were assessed for redness and swelling 24 and 48 hours after removal of the dressings (using the numerical grading system described below (modified from Kligman A.M., J. Invest. Dermatol. 47, 1966)).
The test sites were re-shaved with an electric razor after the first reading.


OTHER:
The choice of corn oil as vehicle in this test was based on the fact that the test article diluted well in corn oil at the concentrations used.
Challenge controls:
10 females
Positive control substance(s):
yes
Remarks:
formaldehyde; Lamers & Pleuger, 's-Hertogenbosch, The Netherlands; batch no. 919 K12325603; purity 37%

Results and discussion

Positive control results:
A positive control experiment is carried out once a year as a sensitivity check of the test system. The most recent test was carried out in April 1991.
Concentrations selected for this study were:
Intradermal induction: 1% (w/w) in physiological saline.
Epidermal induction: 1% (w/w) in distilled water.
Challenge:
a = 0.5% (w/w) in distilled water.
b = 0.25% (w/w) in distilled water.
c = 0.1% (w/w) in distilled water.
d = distilled water.
Clearly positive results were observed in the experimental animals after the challenge with 0.25% (w/w) formaldehyde in distilled water (see Any other information ...).
The test described above was performed under GLP-conditions with a QA-check.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 % w/w in corn oil
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Remarks:
A few animals also showed scaliness
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25 % w/w in corn oil
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Remarks:
One animal also showed scaliness.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 % (w/w) in corn oil
No. with + reactions:
9
Total no. in group:
17
Clinical observations:
These reactions were characterised by red spots and scaliness.
Remarks on result:
no indication of skin sensitisation
Remarks:
Taking into account the intensity of the responses and comparing these with the reactions seen in the control animals, only one animal showed a positive skin reaction in response to the 5% concentration
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25 % w/w in corn oil
No. with + reactions:
8
Total no. in group:
17
Clinical observations:
These reactions were characterised by red spots and scaliness.
Remarks on result:
no indication of skin sensitisation
Remarks:
Taking into account the intensity of the responses and comparing these with the reactions seen in the control animals, only one animal showed a positive skin reaction in response to the 5% concentration.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 % w/w in corn oil
No. with + reactions:
1
Total no. in group:
17
Clinical observations:
These reactions were characterised by red spots and scaliness
Remarks on result:
no indication of skin sensitisation
Remarks:
Taking into account the intensity of the responses and comparing these with the reactions seen in the control animals, only one animal showed a positive skin reaction in response to the 5% concentration.
Reading:
rechallenge
Group:
positive control
Dose level:
0.5 % w/w in distilled water
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Group:
positive control
Dose level:
0.25 % w/w formaldehyde n distilled water
No. with + reactions:
0
Total no. in group:
12
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Group:
positive control
Dose level:
0.1 % w/w of formaldehyde in distilled water
No. with + reactions:
0
Total no. in group:
8
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Group:
positive control
Dose level:
0 %
No. with + reactions:
0
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

INDUCTION

all experimental animals showed severe erythema and slight to well-defined oedema after the 48 hours occluded epidermal induction exposure. Some experimental animals showed white areas, as a sign of necrosis, on the treated skin.

OBSERVATIONS

In addition to the skin reactions the following observations and data were recorded:

Mortality/Viability/Toxicity                             Once daily

Body Weights                                                    During acclimatisation and at termination of the study.

TOXICITY SYMPTOMS / MORTALITY

Two experimental animals were found dead on days 10 and 11, and one animal was killed in extremis for humane reasons on day 14.

In the morning of day 11, it was noted that the experimental animals had eaten little or no food.

On day 13, the day before death, animal 197 showed hypopnoea, piloerection, half closed eyes and emaciation. This animal was therefore killed in extremis on day 14.

At necropsy, the two found dead animals showed red coloured liquid in the abdominal cavity. The animal killed in extremis showed red discoloured small intestines and a yellow discoloured area on the lateral liver lobe at necropsy.

BODY WEIGHTS

A difference between the average body weight gain of experimental and control animals was noted (see table 4, Appendix 1). The experimental animals did not gain as much as the control animals, possibly as a result of test substance treatment.

EVALUATION OF RESULTS

The irritation and sensitisation scores and all other observations were recorded on data sheets and transcribed for compilation and analysis.

The results evident in test animals at the challenge application(s) were compared with the results evident in control animals.

Positive skin reactions (grade 1 or more) were considered signs of sensitisation, provided that such reactions were not observed in the control group.

The readings after the challenge applications were compared to assess the sensitisation rate (%) i.e. the number of sensitised animals in proportion to the total number of animals of the experimental group at the same test substance concentration.

Rating of allergenicity:

Based upon the highest percentage of animals sensitised, the test substancewas assigned to the following classification of Kligman (3.Invest. Dermatol. 47, 393-409, 1966).

classification

% sensitized animals

I. weak

0 – 8%

II. mild

9 – 28%

III. moderate

29 – 64%

IV. strong

65 – 80%

V. extreme

81 – 100%

POSITIVE SKIN REACTIONS TO THE CHALLENGE

Formaldehyde Concentration

0.5%

0.25%

0.1%

0%

EXP. GROUP

animals - positive reaction

20

12

8

0

Sensitisation rate

- (a)

60

40

0

CONTROL GROUP

animals - positive reaction

4

0

0

0

(a) = As the result of similar skin reactions observed in the experimental and control animals in response to this concentration, no sensitisation rate was calculated.

Reference: "Allergic Contact Dermatitis in the Guinea Pig: Identification of Contact Allergens" Magnusson B. Kligman A.M., 1970 published by C.C. Thomas, Springfield, Illinois, U.S.A.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
These results lead to a sensitisation rate of 5%, which indicates that POLYCAT 12 has weak sensitizing properties in this test applying the rating of allergenicity described by Kligman A.M. (1966).

According to the EEC criteria for classification and labelling requirements for dangerous substances and preparations (EEC Directive 91/325/EEC, Amendment to Annex VI of the EEC Directive 67/548/EEC), POLYCAT 12 need not be labelled as a skin sensitiser.
Executive summary:

This study was entitled "Assessment for Contact Hypersensitivity to POLYCAT 12 in the Albino Guinea Pig (Maximization Test)".

The purpose of the study was to obtain information on the potential of POLYCAT 12 to induce delayed contact hypersensitivity (skin sensitisation) in the guinea pig after intradermal and epidermal exposures.

This study was carried out in accordance with the OECD Guideline No. 406, “Skin Sensitisation”, the EEC Directive 84/449/EEC, Part 8.6, “Skin Sensitisation" and in accordance with the method described by Magnusson and Kligman, “Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens”.

After identification of the slightly irritating and the non-irritating test article concentrations in the primary irritation experiments, a main study was performed with the selected test article concentrations. The experimental animals were intradermally injected with a 5 % concentration and epidermally exposed to the undiluted test substance while the control animals were similarly treated, but with the vehicle only. Immediately after the epidermal exposure, the skin irritation was scored. Two weeks after the epidermal application all animals were challenged with test article concentrations of 50 %, 25 % and 10 %, and the vehicle. The challenge reactions were assessed 24 and 48 hours after bandage removal.

The epidermal exposure of POLYCAT 12 in the induction phase resulted in severeskin irritation.The epidermal exposure of POLYCAT 12 in the challenge phase resulted in one positive sensitisation reaction in response to the 10 % test article concentration.

Three experimental animals were found dead or were killed in extremis on days 10, 11 and 14.

Under the conditions used in this study, POLYCAT 12 resulted in a sensitisation rate of 5 %.

Applying the rating of allergenicity described by Kligman A.M. (1966) on the results obtained in this test, POLYCAT 12 is considered to have weak sensitising properties.

Based on these results and according to the EEC criteria for classification and labelling requirements for dangerous substances and preparations (EEC Directive 91/325/EEC, Amendment to Annex VI of the EEC Directive 67/548/EEC), POLYCAT 12 need not be labelled as a skin sensitiser.