Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 January 1981 - 10 April 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed in accordance with generally accepted scientific principles with the experimental methodology being reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Recognised Guinea Pig maximisation test

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Available study data +12 years old

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-Ethylhexyl N-(5-isocyanato-2-methylphenyl)carbamate and 2-Ethylhexyl N-(3-isocyanato-4-methylphenyl)carbamate and N,N'-(4-Methyl-1,3-phenylene)bis(carbamic acid) C,C'-bis(2-ethylhexyl) ester

EC Number:

937-955-6

Molecular formula:

components with the molecular formulae C25H42N2O4 and C17H24N2O3

IUPAC Name:

Reaction mass of 2-Ethylhexyl N-(5-isocyanato-2-methylphenyl)carbamate and 2-Ethylhexyl N-(3-isocyanato-4-methylphenyl)carbamate and N,N'-(4-Methyl-1,3-phenylene)bis(carbamic acid) C,C'-bis(2-ethylhexyl) ester

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Hilltop Lab Animals, Inc.
- Weight at study initiation: 264 - 317 g.
- Diet (e.g. ad libitum): Purina Certified Guinea Pig Chow ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 6 days.


IN-LIFE DATES: From: To: 19 January 1981 - 10 April 1981

First immunizing dose: 19/1/81
Last immunizing dose: 6/2/81

Challenge doses:
23/2/81 Topical - shoulder area
2/3/81 Topical - shoulder area
5/3/81 Inhalation
9/4/81 Topical - shoulder area (25 % dilution in mineral oil)

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: Topical on abraded skin

Vehicle:

unchanged (no vehicle)

Induction 2

Concentration / amount:

0.1 mL

Challenge

Challenge 1

Route:

other: Topical on abraded skin and inhalation

Vehicle:

unchanged (no vehicle)

Challenge 2

Concentration / amount:

0.1 mL

No. of animals per dose:

20

Details on study design:

PRE-SENSITISATION SKIN PENETRATION - SINGLE DOSE TO GUINEA PIGS
Five female guinea pigs were dosed with 8.0 mL/kg of the undiluted sample to determine whether or not the total amount of sample to be administered during a sensitisation study would cause excessive ill-effects. The dorsal and ventral lumbar area was shaved and wrapped in impervious polyethylene sheeting. Sample administered underneath sheeting via syringe and ball-tipped intubation needle. Guinea pigs restrained for exposure period of six hours.
All five guinea pigs survived the 15 day observation period with no signs of distress and nothing remarkable was observed at necropsy. Weight gains were variable, which is considered normal for fully-grown animals such as those used in this study. LD50 >8.0 mL/kg; dosed undiluted.
The five guinea pigs that were utilised for this test had been used three months earlier in a sensitisation study of an unrelated compound, but this prior exposure is not believed to have influenced the results of the present study.

SENSITISATION STUDIES
Preliminary Procedure
A skin irritation test was performed using guinea pigs from a previous negative sensitisation test with a chemically unrelated material to determine if the sample would cause irritation if applied undiluted. Over a period of 5 consecutive days the following procedures were used:
Day 1
The dorsal areas of 3 pigs were clipped as closely as possible using Oster small animal clippers with an ANGRA No. 2 head. This area was abraded using a suede shoe brush by running three strokes parallel with the back of the animal and two more perpendicular to this line on the same minimal 4 square centimetre area. A dose of 0.1 mL of the undiluted sample was rubbed into this area on the abraded skin. A two-ply piece of one-inch square gauze was placed over the dosed area and a second 0.1 mL of the undiluted sample was placed on the gauze. Small ¾ inch diameter plastic caps were placed over the gauze to occlude the dosed area and were held in place with a VINYLITE tie. The pigs were immobilized in restrainers for a six-hour exposure period.

Day 2
A dose of 0.1 mL of the undiluted sample was applied to the same area as was dosed on Day One. The dosed areas were not abraded and the animals were returned to their home cages immediately after dosing.

Day 3
The animals were restrained and 0.1 mL of the undiluted sample was applied three times at three hour intervals to the same area as was previously dosed. The dosed areas were not abraded.

Day 4
The dosed areas were re-clipped and the same procedure as Day One was followed except the dosed areas were not abraded.

Day 5
The same procedure as Day One was followed except the animals were not clipped.

The skin irritation resulting from the above procedures was moderate capillary injection at Days 3, 4, 5, and 6. On the basis of these results, it was decided to use the undiluted sample for the sensitisation test.

MAIN TEST
The sensitisation regimen consists of nine initial 6 hour contact periods over three weeks, a two-week incubation (rest) period, and a challenge dose. The test was initiated 19/1/1981.

SENSITISATION PROCEDURE
Initial
The hair on the dorsal area of 20 guinea pigs was clipped as closely as possible using Oster small animal clippers with an ANGRA No. 2 head. This area was then abraded using a suede shoe brush. The method of abrading was three strokes parallel with the back of the animal and two more perpendicular to this line on the same minimal 4 cm2 area. After placing the pigs in restrainers, a dose of 0.1 mL of the undiluted sample was rubbed into a small area on the abraded skin. Two plies of one-inch square gauze were placed over the dosed area and a second 0.1 mL of the undiluted sample was placed on the gauze. Small ¾ inch diameter by ½ inch deep plastic caps were placed over the gauze to occlude the dosed area and held in place with a VINYLITE tie. The animals were immobilised in the restrainers for a six-hour exposure period. The above procedure was followed for the nine initial applications except that clipping was performed only as necessary to insure good contact of the undiluted sample to the skin. Throughout the nine applications, the dosed area on the dorsum was varied so that irritation from repeated application to the same area would not develop.

Challenge
After a two-week rest or incubation period on 23/2/1981, the animals were challenged using undiluted sample. The hair was closely clipped from the upper back and both shoulder blades of all the guinea pigs. After restraining them, a dose of 0.1 mL of the undiluted sample was rubbed into the intact skin of the left shoulder blade. The right shoulder blade was used as a control to facilitate reading the reactions. Two plies of one-inch square gauze were placed over the dosed area and a VINYLITE tie was placed around the restrainer to hold the gauze in place and to occlude the dosed area. To avoid pressure irritation, caps were not placed over the gauze during the challenge.
The pigs were kept in restraint for a period of six hours. Upon removal from the restrainers, any reactions were noted and recorded. Twenty-four and forty-eight hours later reactions were noted and recorded.

One week after the challenge, the animals were re-challenged on the left shoulder blade using the right shoulder blade as a control to facilitate reading the reactions. A dose of 0.1 mL of the undiluted sample was applied to the intact skin of the left shoulder blade and the animlals returned to their cages immediately after application. Reactions were noted and recorded six hours later. Twenty-four and forty-eight hours later any reactions were noted and recorded .

Inhalation Challenge
An inhalation challenge was performed three days after the re-challenge on 5/3/1981. Ten of the guinea pigs were placed in a 360 litre inhalation chamber at the same time the undiluted sample was introduced. The sample was magnetically stirred throughout the exposure period to facilitate the release of vapours into the chamber. After two hours, the first ten were removed from the chambers and the second ten guinea pigs were introduced to the same chamber with the sample stirred throughout the two-hour exposure period. At twenty-four hours after exposure, all the pigs were checked for eye irritation by staining for 20 seconds with 5 % aqueous fluorescein solution.

Re-challenge
Prior to using 25 % concentration in mineral oil as a challenge, the guinea pigs were subjected to one dose of 0.01 mL of undiluted sample and of 1, 5 and 10 % dilutions in mineral oil at 2-day intervals.

On 9/4/1981, a third topical challenge was performed on the animals using a 25 % dilution of the sample in mineral oil. The hair on the upper back and both shoulder blades was clipped as closely as possible using small animal clippers. A dose of 0.1 mL of the 25 % dilution in mineral oil was placed on the left shoulder blade and a dose of 0.1 mL of undiluted mineral oil was placed on the right shoulder blade. The guinea pigs were returned to their cages immediately after dosing. Reactions were noted and recorded at six and twenty-four hours after dosing.

Reading the Reactions
The following system adopted from the Chemical Hygiene Fellowship of Mellon Institute was used to read the reactions:

0 - no reaction
1 - very faint pink (vfp) - no value is recorded
2 - faint pink (fp)
3 - pink (p)
4 - red (r)
5 - bright red (R)
6 - oedema < 1 mm in height (o)
7 - oedema > I mm in height (O)
8 - necrosis (nec)

Positive control substance(s):

not specified

Results and discussion

Positive control results:

Not specified

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

undiluted

No. with + reactions:

4

Total no. in group:

20

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

undiluted

No. with + reactions:

0

Total no. in group:

20

Results 3

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

undiluted

No. with + reactions:

5

Total no. in group:

20

Results 4

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

undiluted

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Not specified

Remarks on result:

no indication of skin sensitisation

Results 5

Key result

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

The right shoulder blade was used as a control to facilitate reading the reactions to which no reactions were observed

Remarks on result:

no indication of skin sensitisation

Results 6

Key result

Group:

positive control

Dose level:

Not specified

No. with + reactions:

0

Total no. in group:

0

Clinical observations:

Not specified

Remarks on result:

other: Not specified

Any other information on results incl. tables

Table 1 Results of the First Challenge - 6 hour Reading (23/2/81) and 24 and 48 hour Reading (24/2/81 and 25/2/81)

Topical Application (semi-occluded)

Animal Number	Weight (g)		Prechallenge Reading (G1)	6 Hour Reading	Area Involved (mm)	24 Hour Reading (G2)	Area Involved (mm)	48 Hour Reading (G3)	Grade
	Initial (19/1/81)	Final (23/2/81)							24	48
P-2627	271	456	0	fp	23 x 30	0	-	0	0	0
P-2628	290	500	0	fp	25 x 35	0	-	0	0	0
P-2629	271	612	0	vfp	32 x 28	0	-	0	0	0
P-2630	317	537	0	vfp	23 x 28	0	-	0	0	0
P-2631	269	455	0	fp	25 x 25	0	-	0	0	0
P-2632	310	553	0	fp	27 x 29	0	-	0	0	0
P-2633	264	433	0	vfp	30 x 30	vfp	10 x 15	0	0	0
P-2634	280	436	0	fp	30 x 32	0	-	0	0	0
P-2635	297	538	0	fp	32 x 25	0	-	0	0	0
P-2636	293	507	0	fp	25 x 30	vfp	10 x 10	0	0	0
P-2637	279	529	0	vfp	24 x 31	0	-	0	0	0
P-2638	305	545	0	p	28 x 32	0	-	0	0	0
P-2639	286	514	0	p	27 x 33	0	-	0	0	0
P-2640	275	492	0	vfp	23 x 31	0	-	0	0	0
P-2641	311	553	0	p	25 x 25	0	-	0	0	0
P-2642	283	522	0	fp	30 x 30	0	-	0	0	0
P-2643	292	498	0	fp	30 x 32	vfp	10 x 10	0	0	0
P-2644	291	495	0	fp	30 x 30	0	-	0	0	0
P-2645	287	460	0	vfp	27 x 30	0	-	0	0	0
P-2646	283	463	0	fp	28 x 30	vfp	15 x 15	0	0	0

 

Table 2 Results of the Second Challenge - 6 hour Reading (2/3/81) and 24 and 48 hour Reading (3/3/81 and 4/3/81)

Topical Application (not occluded)

Animal Number	Weight (g)		Prechallenge Reading (G1)	6 Hour Reading	Area Involved (mm)	24 Hour Reading (G2)	Area Involved (mm)	48 Hour Reading (G3)	Grade
	Initial (19/1/81)	Final (23/2/81)							24	48
P-2627	271	470	0	fp	25 x 20	0	-	0	0	0
P-2628	290	544	0	fp	28 x 30	0	-	0	0	0
P-2629	271	657	0	fp	33 x 25	0	-	0	0	0
P-2630	317	575	0	vfp	28 x 30	0	-	0	0	0
P-2631	269	478	0	p	30 x 27	vfp	27 x 30	0	0	0
P-2632	310	559	0	fp	30 x 26	0	-	0	0	0
P-2633	264	461	0	fp	30 x 27	0	-	0	0	0
P-2634	280	472	0	p	30 x 30	vfp	30 x 30	0	0	0
P-2635	297	548	0	fp	30 x 30	0	-	0	0	0
P-2636	293	522	0	fp	30 x 30	0	-	0	0	0
P-2637	279	576	0	fp	30 x 27	0	-	0	0	0
P-2638	305	574	0	p	30 x 30	vfp	30 x 30	0	0	0
P-2639	286	530	0	fp	30 x 30	0	-	0	0	0
P-2640	275	538	0	fp	25 x 28	0	-	0	0	0
P-2641	311	602	0	p	30 x 30	vfp	30 x 30	0	0	0
P-2642	283	545	0	p	30 x 30	0	-	0	0	0
P-2643	292	517	0	fp	30 x 28	0	-	0	0	0
P-2644	291	502	0	vfp	25 x 30	0	-	0	0	0
P-2645	287	480	0	fp	30 x 25	0	-	0	0	0
P-2646	283	453	0	p	25 x 30	vfp	25 x 30	0	0	0

 

Table 3 Results of the Third Challenge - 6 hour Reading (9/4/81) and 24 hour Reading (10/4/81)

Topical Application (not occluded, 25 % in mineral oil)

Animal Number	Prechallenge Reading (G1)	6 Hour Reading	Area Involved (mm)	24 Hour Reading (G2)	Area Involved (mm)	Grade
						24
P-2627	0	vfp	25 x 28	0	-	0
P-2628	0	vfp	20 x 25	fp	25 x 30	0
P-2629	0	vfp	25 x 30	0	-	0
P-2630	0	fp	30 x 30	0	-	0
P-2631	0	vfp	20 x 25	vfp	20 x 20	0
P-2632	0	fp	25 x 20	vfp	25 x 20	0
P-2633	0	vfp	30 x 20	vfp	25 x 20	0
P-2634	0	vfp	25 x 20	0	-	0
P-2635	0	vfp	15 x 25	vfp	20 x 20	0
P-2636	0	vfp	20 x 30	0	-	0
P-2637	0	vfp	20 x 25	0	-	0
P-2638	0	fp	30 x 30	vfp	25 x 20	0
P-2639	0	vfp	25 x 30	0	-	0
P-2640	0	fp	30 x 30	0	-	0
P-2641	0	fp	25 x 30	0	-	0
P-2642	0	p	15 x 25	vfp	20 x 15	0
P-2643	0	vfp	25 x 20	0	-	0
P-2644	0	fp	25 x 25	vfp	20 x 20	0
P-2645	0	0	-	0	-	0
P-2646	0	vfp	25 x 25	0	-	0

The mineral oil control areas had no reactions at the prechallenge or the 6 hour readings on any of the guinea pigs.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The increase in reactions seen for each challenge stage of the study would seem to indicate that the test material has sensitising potential.

Executive summary:

Efforts were made to determine the sensitising ability of the test material in guinea pigs. A topical application route developed in the testing laboratory was utilised, with an additional inhalation challenge and a retest topical challenge with a 25 % dilution of sample in mineral oil.

 

On the basis of the 4 or 6 hour skin responses which reflect circulatory antibodies, the 20 female guinea pigs responded to the undiluted challenge application with 6 faint and 14 solid reactions. A week later upon similar rechallenge, there were only 2 faint or minimal reactions and 18 solid reactors. When challenged by a 2 hour exposure to substantially saturated vapour 3 days later, the only detectable response was nasal discharge from 3 animals. The relationship of this result to treatment cannot be evaluated, but no asthmatic responses could be detected. A month later, after checking responses to low concentrations in mineral oil, the pigs were challenged with a 25 % solution in mineral oil. The 6 hour reading revealed 12 faint reactions and 8 solid reactions.

The 24 hour readings likewise show a gradual build-up of reactors with each successive challenge (excluding inhalation) from 4 to 5 to 8 reactors to the topical challenges. The 24 hour results supposedly reflect cell mediated responses (in the skin) and are responsible for contact dermatitis in humans.