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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation, other
Remarks:
in-vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-03-09 - 2001-12-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Commission Directive 96/54/EC Method B6 Acute Toxicity (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An appropriate guinea pig maximisation test from 2001 is available. Conducting an additional LLNA is not necessary.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Test item: 2,6-DIISOPROPYLPHENYLCARBODIIMIDE
- Substance type: organic technical product
- Description : white solid
- Lot/batch no: 0600181/009
- Date received: 19 July 2001
- Storage conditions: room temperature, in the dark

For the purpose of this study the test material was freshly prepared in arachis oil BP. The concentrations used are discussed in the procedure section.
The absorption of the test material was not determined.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS: Twenty-one male albino Dunkin Hartley guinea pigs
- Source: by David Hall Limited, Burton-on-Trent, Staffordshire, UK.
- Age at study initiation: approximately eight to twelve weeks.
- Weight at study initiation: 300 to 450g
- Housing: singly or in pairs in solid-floor polypropylene cages furnished with woodflakes.
- Diet (e.g. ad libitum): Certified Guinea Pig Diet (Code 5026, supplied by PMI Nutrition International, Nottingham, UK), ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days
- Other a): animal selection at random, unique number within the study was written on a small area of clipped rump using a black indelible marker-pen.
- Other b): The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C,
- Humidity (%): 30 to 70%,
- Air changes (per hr): at least fifteen changes per hour,
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
- Other: Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
arachis oil
Concentration / amount:
Intradermal Induction : 5% w/w in arachis oil BP
Topical Induction : 75% w/w in arachis oil BP
Topical Challenge : 25% and 10% w/w in arachis oil BP
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
arachis oil
Concentration / amount:
Intradermal Induction : 5% w/w in arachis oil BP
Topical Induction : 75% w/w in arachis oil BP
Topical Challenge : 25% and 10% w/w in arachis oil BP
No. of animals per dose:
10 Test animals und 5 control animals
Details on study design:
Induction:
Shortly before treatment on day 0 the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 mL each) was made on each side of the midline into a 20 mm x 40 mm area.
The injections were:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 5% w/w formulation of the test material in arachis oil BP
c) a 5% w/w formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites was evaluated.

On day 7 the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation. A filter paper patch (WHATMAN No. 4: approximate size 40 mm x 20 mm), saturated with the test material formulation (75% w/w in arachis oil BP) was applied to the prepared skin and held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.

The degree of erythema and oedema was quantified 1 and 24 hours following removal of the patches.
Any other reactions were also recorded.

Challenge:
Shortly before treatment on day 21, an area of approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers.
A square filter paper patch (WHATMAN No. 4: approximate size 20 mm x 20 mm), saturated with the test material formulation at the maximum non-irritant concentration (25% w/w in arachis oil BP) was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 10% w/w in arachis oil BP was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal.
After 24 hours, the dressing was carefully removed and discarded. The challenge sites were swabbed with cotton wool soaked in diethyl ether to remove residual material. The position of the treatment sites was identified by using a black indelible marker-pen.
Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.
Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified.
Any other reactions were also recorded.

The method used for assessing the sensitising properties of the test material was based on the Guinea Pig Maximisation Test of Magnusson B & Kligman A M, J. Invest. Dermatol. (1969) 52: 268 - 276.
Challenge controls:
Induction of the Control Animals: The intradermal induction was performed using an identical procedure to that used for the test animals except that the test material was omitted from the intradermal injections. Injection b) was therefore the vehicle alone, injection c) was a 50% formulation of the vehicle in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water. Similarly, the topical induction procedure was identical to that used for the test animals except that the test material was omitted.
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde (Induction: 5 % in arachis oil BP, Topical challenge: 100 % and 75 % w/w in arachis oil BP)

Results and discussion

Positive control results:
of 10 male Test animals: 2 of 10 = 20 % reacted with a sensitisation to the positive control substance

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 % w/w in arachis oil BP
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25 % w/w in arachis oil BP. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10 % w/w in arachis oil BP
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 % w/w in arachis oil BP. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25 % w/w in arachis oil BP
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25 % w/w in arachis oil BP. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10 % w/w in arachis oils BP
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 % w/w in arachis oils BP. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Any other information on results incl. tables

Moderate and confluent erythema was noted at the intradermal induction sites of test group animals.

Discrete or patchy erythema was noted at the intradermal induction sites of control group animals.

Discrete or patchy to moderate and confluent erythema was noted at the topical induction sites of test group animals. Discrete or patchy erythema was noted at the topical induction sites of four control group animals. Bleeding from the intradermal injection sites was noted in seven test group animals and three control group animals. Residual test material was also noted at the topical induction sites of four test group animals.

Body weight increases of the guinea pigs in the test group between day 0 and day 24 were comparable to those noted in the control group animals over the same period.

Table1- Individual Skin Reactions at Challenge
Challenge concentrations: 25 % and 10 % w/w
Vehicle: Arachis Oil BP 
Group Animal Number Skin Reactions (Hours after Removal of Dressings)
24Hours 48Hours
10% 25% 10%  25%
Erythema Oedema Other Erythema Oedema Other Erythema Oedema Other Erythema Oedema Other
Test 1 0 0 - 0 0 - 0 0 - 0 0 -
2 0 0 - 0 0 - 0 0 - 0 0 -
3 0 0 - 0 0 - 0 0 - 0 0 -
4 0 0 - 0 0 - 0 0 - 0 0 -
5 0 0 - 0 0 - 0 0 - 0 0 -
6 0 0 - 0 0 - 0 0 - 0 0 -
7 0 0 - 0 0 - 0 0 - 0 0 -
8 0 0 - 0 0 - 0 0 - 0 0 -
9 0 0 - 0 0 - 0 0 - 0 0 -
10 0 0 - 0 0 - 0 0 - 0 0 -
Control 11 0 0 - 0 0 - 0 0 - 0 0 -
12 0 0 - 0 0 - 0 0 - 0 0 -
13 0 0 - 0 0 - 0 0 - 0 0 -
14 0 0 - 0 0 - 0 0 - 0 0 -
15 0 0 - 0 0 - 0 0 - 0 0 -
- =No other reactions noted

Table 2: Intradermal Induction - Individual Skin Reactions
Group Animal Number Grade of Erythema at Observation Site
24 Hours 48 Hours
Left Side Right Side Left Side Right Side
TEST 1 2 2 2 2
2 2 2 2 2
3 2 2 2 2
4 2 2 2 2
5 2 2 2 2
6 2 2 2 2
7 2 2 2 2
8 2 2 2 2
9 2 2 2 2
10 2 2 2 2
CONTROL 11 1 1 0 0
12 1 1 1 0
13 1 1 0 0
14 1 1 1 1
15 1 1 0 0

Table3: Topical Induction - Individual Skin Reactions
Group Animal Number Skin Reactions (Hours After Removal of Dressing)
1 Hour 24 Hours
Erythema Oedema Other Erythema Oedema Other

TEST

1 2 0 Bs 2 0 -
2 2 0 Bs 2 0 -
3 1 0 - 2 0 -
4 2 0 BsRt 2 0 -
5 2 0 - 1 0 -
6 2 1 - 1 0 -
7 2 1 BsRt 2 0 -
8 2 1 Bs 2 0 -
9 2 1 BsRt 2 0 -
10 2 1 BsRt 2 0 -
CONTROL 11 1 0 Bs 0 0 -
12 1 0 - 0 0 -
13 1 0 - 0 0 -
14 0 0 Bs 0 0 -
15 1 0 Bs 0 0 -

Er = Erythema

Oe = Oedema

Bs = Bleeding from the intradermal injection site

- = No other reactions noted

Rt = Residual test material

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: EU-GHS
Conclusions:
The study was performed according to the OECD Guideline 406 with no deviations and according to the good laboratory practice principles, it is considered to be of the highest quality (reliability Klimisch 1). The criteria of validity of the test system are fulfilled. The test material did not induce a sensitisation on the intact skin of guinea pigs. The test material was considered to be not sensitising under the conditions of the test.
Under the conditions of the test, the test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitizer to guinea pig skin.
The test material did not meet the criteria for classification as a sensitizer according to the European regulation (EC) No. 1272/2008. No symbol and risk phrase are required.
Executive summary:

The skin sensitisation potential of the test substance was investigated in guinea pigs according to OECD TG406 (Driscoll, 2001). The test substance (5 % in Arachis Oil BP) was injected intradermally for induction (3 consecutive applications). For topical induction a test substance solution of 75% w/w was used. For challenge a patch moistened with a 25 or 10% solution of the test material dissolved in Arachis Oil BP was applied to the shorn skin of the guinea pigs for 24 hours. No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48-hour observations with 25% w/w in Arachis Oil BP. Additionally no skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 4 8-hour observations with 10% w/w in Arachis Oil BP. Therefore the substance is not considered to be a skin sensitizer.