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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study have been performed before the REACh legislation enters into force.

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-Methylenediphenyl diisocyanate, oligomeric reaction products with 2,4'-diisocyanatodiphenylmethane and oxydipropanol
EC Number:
500-270-4
EC Name:
4,4'-Methylenediphenyl diisocyanate, oligomeric reaction products with 2,4'-diisocyanatodiphenylmethane and oxydipropanol
Molecular formula:
C14 H10 N O [C21 H24 N2 O5 ]n N C Onmean = 1, 2, 3...
IUPAC Name:
Oligomeric reaction product of 1,1'-methylenebis(4-isocyanatobenzene) and oxybispropanol
Constituent 2
Reference substance name:
propane-1,2-diol polymer with 1-isocyanato-4-[(4- isocyanatophenyl)methyl]benzene and 1-isocyanato-2-[(4- isocyanatophenyl)methyl]benzene
IUPAC Name:
propane-1,2-diol polymer with 1-isocyanato-4-[(4- isocyanatophenyl)methyl]benzene and 1-isocyanato-2-[(4- isocyanatophenyl)methyl]benzene

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Total Number: 9 Dose Range-finding
10 Test Article Group
5 Vehicle Control Group
Gender: Male and/or female. Females were nulliparous and nonpregnant.
Age Range: Dose Range: 4-5 weeks, Young adult
Main Study: 6 weeks, young adult at the start of dosing for the main study. Records of dates of birth for animals used in this study are retained in the Calvert archives.
Body Weight Range: Dose Range Study: 329 to 434 grams
Main Study: 364 to 481 grams at the outset (Day 1) of the study.
Animal Source: Elm Hill Breeding Laboratories, Chelmsford, MA 01824
Experimental History: Purpose-bred and experimentally naïve at the outset of the study.
Identification: Ear tag and cage card

ENVIRONMENTAL CONDITIONS
Housing: Animals were individually housed in compliance with USDA Guidelines. The room in which the animals were kept was documented in the study records. No other species were kept in the same room. Enrichment was provided as per Calvert SOP.
Lighting: 12 hours light/12 hours dark
Room Temperature: 19 to 26°
Relative Humidity: 20 to 83%
Food: All animals had access to Harlan Teklad Guinea Pig Diet (certified) ad libitum. The lot number(s) and specifications of each lot used are archived at Calvert. No contaminants were known to be present in the certified diet at levels that would be expected to interfere with the results of this study. Analysis of the diet was limited to that performed by the manufacturer, records of which are maintained in the Calvert archives.
Water: Tap water was available ad libitum, to each animal via an automatic watering device. The water is routinely analyzed for contaminants as per Calvert SOP’s. No contaminants were known to be present in the water at levels that would be expected to interfere with the results of this study. Results of the water analysis are maintained in the Calvert archives.
Acclimation: Study animals were acclimated to their housing for a minimum of 7 days prior to their first day of dosing.

IN-LIFE DATES: From: 31 May 2009 To: 9 Jul 2009

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Jeffsol - Propylene Carbonate - NF
Concentration / amount:
It was important to use an 'anhydrous' or low moisture vehicle in order to prevent hydrolysis of MDI to MDA. Therefore no preparations of the test article were made in conjunction with water. All preparations including the test article were made in the Sponsor’s vehicle (Jeffsol - Propylene Carbonate - NF).
The test article was prepared at 0.5% (v/v) in Jeffsol Propylene Carbonate - NF and FCA/Jeffsol - Propylene Carbonate - NF for intradermal induction.For topical induction and challenge the test article was prepared at 75% and 25% in Jeffsol-Propylene Carbonate - NF, respectively. The test article bottle was purged with nitrogen after use.
Dosing preparations were stored at room temperature following preparation.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Jeffsol - Propylene Carbonate - NF
Concentration / amount:
It was important to use an 'anhydrous' or low moisture vehicle in order to prevent hydrolysis of MDI to MDA. Therefore no preparations of the test article were made in conjunction with water. All preparations including the test article were made in the Sponsor’s vehicle (Jeffsol - Propylene Carbonate - NF).
The test article was prepared at 0.5% (v/v) in Jeffsol Propylene Carbonate - NF and FCA/Jeffsol - Propylene Carbonate - NF for intradermal induction.For topical induction and challenge the test article was prepared at 75% and 25% in Jeffsol-Propylene Carbonate - NF, respectively. The test article bottle was purged with nitrogen after use.
Dosing preparations were stored at room temperature following preparation.
No. of animals per dose:
10
Details on study design:
The test item was evaluated in the Magnusson-Kligman guinea pig maximization model of delayed hypersensitivity. The main study was conducted with 10 animals for the test article group and 5 animals for the vehicle control group. The positive control (DNCB) group was run under an in-house Calvert study 0423GC62.010. Induction doses of 0.5% and 75% were used intradermally and topically, respectively. On Day 22, animals were dermally challenged with the test article at naïve sites at a dose of 25%.

During the intradermal induction procedure, FCA was employed as part of the dose preparation procedure. The test article was prepared at 0.5% (v/v) in Jeffsol Propylene Carbonate - NF and FCA/Jeffsol-Propylene Carbonate - NF for intradermal induction. For topical induction and challenge the test article was prepared at 75% and 25% in Jeffsol-Propylene Carbonate - NF, respectively. The test article bottle was purged with nitrogen after use.
Dosing preparations were stored at room temperature following preparation.

The dose levels were based upon the results of a dose-range finding study. Prior to dose administration, the Study Director in agreement with the Study Monitor authorized the choice of the dose levels for the definitive study. The test article concentration selected for intradermal induction was 0.5% and topical was 75%. The challenge dose was the highest non-irritating concentration (HNIC) of the test article which was determined to be 25%.

Volume Administration:
0.1 mL - intradermal per injection site
0.3 mL - topical induction per site
0.2 mL - topical challenge per site

Route: Intradermal - First Induction
Dermal - Second Induction and Challenge
Frequency: Intradermal Induction - First Week (Day 1)
Dermal Induction - Second Week (Day 8)
Dermal Challenge - Fourth Week (Day 22) (i.e. 15 days after dermal induction)

Site Preparation and Intradermal Induction Stage:
The hair from an area over the shoulders of the guinea pigs was removed 24 to 48 hours prior to dosing using a clipper. The test article and vehicle control groups with and without FCA were injected in the shoulder region of each animal. A row of three injections, on each side of the animals, (i.e., 6 in all), was made as follows:
(1 & 2) 0.1 mL of FCA (1:1 mixture with distilled water)
(3 & 4) 0.1 mL of the test article in the vehicle (0.5% v/v) or the vehicle (Jeffsol Propylene Carbonate - NF) alone
(5 & 6) 0.1 mL of the test article in FCA (1:1) at 0.5% v/v or
the vehicle emulsified with FCA and the vehicle (Jeffsol-Propylene Carbonate - NF) (1:1)

Injections 1 & 2 were given close to each other nearest to the head and injection 5 & 6 are given most caudally; injections 3 & 4 are given between injection sites 1 & 2 and 5 & 6. The injection sites were just within the boundaries of a 2 x 4 cm patch, which was applied one week later for the topical induction.

Topical Induction Stage: After six days, the test sites were reclipped free of hair. Since irritation was observed in the animals dosed with the test article during the topical range-finding study, no pretreatment was performed.
One week after intradermal injections (Day 8), 0.3 mL of the test article (75%) was spread over a 2 x 4 cm filter paper and applied to the injection site area and occluded using Blenderm® tape.
The Blenderm® tape was held in place with an appropriate bandage. A minimum of forty eight hours later the dressings were removed. This same procedure was employed with the vehicle control article (0.3 mL) in the vehicle control animals. After the 48-hour exposure period, the induction sites were unwrapped and any residual material removed with gauze.

Challenge Period: On Day 22, the test article group and vehicle control group animals were challenged with occluded patches of the test article (25%) and vehicle for 24 hours on the left and right flanks, respectively. The hair was removed from these areas using a clipper 24 hours before application of the patches on Day 21. A 2 x 2 cm filter paper was saturated (0.2 mL) with the test article (25%) and applied to the left flank. Another 2 x 2 cm filter paper was saturated (0.2 mL) with the vehicle (Jeffsol-Propylene Carbonate - NF) and applied to the right flank. The same occlusive technique as for topical induction was employed. Approximately twenty four hours later, the wrapping was removed and the sites wiped clean with gauze. Twenty one hours after unwrapping, the challenge sites were depilated with Nair Lotion Hair Remover (lot # LL805B). Three hours later the sites were graded for elicited skin reactions (24-hour grade). Approximately twenty-four hours later the sites were graded a second time (48-hour grade).
Challenge controls:
Vehicle and Positive controls
Positive control substance(s):
yes
Remarks:
1-chloro-2, 4-dinitrobenzene (DNCB)

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 10.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 10.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
1
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 1.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
1
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 1.0. Total no. in groups: 5.0.

Any other information on results incl. tables

No mortality was observed in any vehicle control or test article group animals on study. No clinical signs were observed in any vehicle or test article group animals on study.

Incidence of Dermal Irritation Scores at Challenge

 

 

Dermal Irritation Scores

 

Induction

Challenge

24-hour Grade

48-hour Grade

 

Treatment

Treatment

0

1

2

3

4

0

1

2

3

4

 

Vehicle Control

Test Item

@25%

4

0

1

0

0

4

0

1

0

0

Jeffsol Propylene Carbonate-NF

5

0

0

0

0

5

0

0

0

0

Test
Article

Test Item

@ 25%

0

0

0

8

2

0

0

1

8

1

Jeffsol Propylene Carbonate-NF

7

1

2

0

0

8

2

0

0

0

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Under the conditions of this study, an intradermal induction of the test item at 0.5% with a topical induction at 75%, followed by a topical challenge at 25% to guinea pigs did elicit a dermal sensitization response at 24 or 48 hours post treatment. Therefore, the test item is considered to be a contact sensitizer in Guinea Pigs.
Executive summary:

The purpose of this study was to determine if the test article elicits a delayed dermal contact hypersensitivity response in guinea pigs.The test item was evaluated in the Magnusson-Kligman guinea pig maximization model of delayed hypersensitivity. Prior to experimental initiation of the induction, the irritation potential of the test article was determined with a dose range-finding studies (intradermal and topical) utilizing 8 naïve guinea pigs. Based upon these results, the intradermal dose utilized was 0.5%, the topical induction dose was 75% and the challenge dose utilized was 25% in the main study.

Two weeks after the topical induction, the hair was removed from right and left flanks. On Day 22, all test article and vehicle control group animals were challenged with occluded patches for 24 hours on the left and right flanks. A 2 x 2 cm filter paper was saturated (0.2 mL test article) with the test article at 25% and applied to the animal’s left flank. Another 2 x 2 cm filter paper was saturated (0.2 mL) with the vehicle (Jeffsol Propylene Carbonate-NF) and applied to the animal’s right flank. The same occlusive technique was employed as for topical induction. After 24 hours, sites were unwrapped and wiped cleaned with gauze. Twenty one hours after unwrapping, the sites were depilated. Three hours later the sites were graded for elicited skin reactions (24-hour grade). Approximately 24 hours later the sites were graded a second time (48-hour grade). 

The main study was conducted with 10 animals for the test article group and five animals for the vehicle control group. Positive control information is provided in a positive control study report.

For the intradermal induction phase (Day 1), each guinea pig received intradermal injections (0.1 mL each) of Freund’s Complete Adjuvant and either Jeffsol Propylene Carbonate-NF (vehicle control group) or the test article at a concentration of 5% (test article group) with and without Freund’s Complete Adjuvant for a total of six dose sites.

On Day 8, either the test article at 75% (test article group-0.3 mL) or Jeffsol Propylene Carbonate-NF (vehicle control group-0.3 mL) was spread over a 2 x 4 cm filter paper and applied to the injection site areas. Blenderm® tape was used to occlude the injection area. The dressings were removed following 48 hours of exposure.

An intradermal induction of test item at 0.5% with a topical induction at 75%, followed by a topical challenge at 25% to guinea pigs elicited a dermal sensitization response at 24 or 48 hours post treatment in 10 of 10 animals.

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