4,4'-Methylene-bis-(3-chloro-2,6-diethylaniline)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The maximization test on guinea pigs was performed for new substance notification (ELINCS) in 1986, before the LLNA testing method was available.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

402-130-7

EC Name:

-

Cas Number:

106246-33-7

Molecular formula:

C21 H28 Cl2 N2

IUPAC Name:

4-[(4-amino-2-chloro-3,5-diethylphenyl)methyl]-3-chloro-2,6-diethylaniline

Test material form:

other: solidified melt

Details on test material:

- Substance code: P5367
- Batch No.: 1/86
- Appearance: Brown solidified melt
- Storage: At ambient temperature in the dark

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Sex: 40 females (20 test group/20 control group)
- Source: David Hall Ltd.
- Weight at study initiation: 300 - 350 g
- Housing: gang housingf (5 animals/cage) in aluminium cages
- Diet: Special Diet Services FD1 Guinea Pigs Diet
- Water: ad libitum
- Acclimatisation: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 23°C
- Humidity: mean 52%

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

paraffin oil

Concentration / amount:

10% w/w

Day(s)/duration:

epicutaneous application six days after intradermal treatment

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

25% w/w

Day(s)/duration:

24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Number of animals in test group: 20
Number of animals in negative control group: 20

Details on study design:

RANGE FINDING TESTS:
A preliminary range finding test was carried out to assess suitable concentrations for injection and topical application on 4 previously untreated guinea pigs. Injections were in the shaved scapular region and topical application was on the shaved flanks. Two guinea pigs were subjected to intradermal injections and a further 2 guinea pigs were exposed to topical application of the test substance at various concentrations.
Concentrations investigated were:
- Injections: 10%, 5%, 2% and 1% w/v in paraffin oil
- Topical application: 25%, 10%, 5% and 2% w/v in paraffin oil

MAIN STUDY:

1. Injection Phase
In the main test, hair was shaved from an area 4 em x 6 em across the scapular region of 42 guinea pigs. These animals were allocated as follows:
- Test group: 20 guinea pigs
- Control group: 20 guinea pigs
- Dose ranging: 2 guinea pigs
The test group guinea pigs were each given 6 intradermal injections, 3 in a line each side of and parallel to the mid-line in the shaved region as follows:
- 0.10 ml Freund's Complete Adjuvant (anterior injection)
- 0.10 ml test material (middle injection)
- 0.05 ml test material emulsified with 0.05 ml Freund's Complete Adjuvant (posterior injection)
The test material was injected at a concentration of 10% w/v in paraffin oil. The 20 control group guinea pigs were similarly treated but with paraffin oil replacing the test material. Twenty four hours after injection the treated sites of both test and control groups were assessed for irritancy. The dose ranging guinea pigs were each given a 0.10 ml intradermal injection of Freund's Complete Adjuvant on either side of the mid-line of the shaved scapular region

2. Topical Application
Six days after the injection phase, the injection site of each of the test and control group animals was shaved again and then wetted with 10% aqueous SLS to provoke a mild inflammatory response to enhance the possibility of sensitisation. After 24 h, a 2 cm x 4 cm patch of Whatman No. 3MM filter paper, charged with the test material at a concentration of 10% w/v in paraffin oil, was applied to the pretreated area of each of the test group animals and the patch covered by an overlapping patch of impermeable plastic adhesive tape (Blenderm). The whole area was then firmly bound with Sleek occlusive tape, and the dressing was left in place for 48 h before removal. The control group guinea pigs were similarly treated but with paraffin oil replacing the test material. Twenty four hours after patch removal the treated sites of both test and control groups were assessed for irritancy. The 2 dose ranging guinea pigs were untreated at this stage.

CHALLENGE EXPOSURE:

1. Dose Ranging for Challenge
Before challenging the test animals, a maximum non-irritant concentration of the test material was determined in a preliminary experiment of 2 dose ranging guinea pigs. The animals were pretreated at the induction phase with Freunds Complete Adjuvant only. The test material was applied to the shaved flanks of the guinea pigs for 24 h at concentrations of 10% and 5% w/v in paraffin oil under the same system used for the topical induction.

2. Challenge by Topical Application
Two weeks after the topical induction, both the test and control group guinea pigs were challenged with the test material at a concentration of 25% w/v paraffin oil and the vehicle. A 5 cm x 5 cm area of the left flank was shaved and the test and control materials were applied to the prepared test site on 2 cm x 2 cm pieces of filter paper. The patches were held in place for 24 h by the same method employed for the topical induction after which time they were removed.

Assessment of Response
The degree of response was determined by trained assessors 24 h and 48 h after removal of the challenge patch when an allergic response would be at a peak. Any animal showing erythema at the site of challenge was considered to have shown a positive response. Reactions are scored according to the following scale:
- No visible change: 0
- Slight or discrete erythema: 1
- Moderate and confluent erythema: 2
- Intense erythema and swelling: 3

The sensitisation potential of the test material is rated according to the percentage of animals sensitised, based on Magnusson-Kligman as follows (snjsitizuation grade):
- 0-8%: Grade I (weak)
- 9-28%: Grade II (mild)
- 29-64%: Grade III (moderate)
- 65-80%: Grade IV (strong)
- 81-100%: Grade V (extreme)

Challenge controls:

Paraffine oil only

Positive control substance(s):

no

Results and discussion

Positive control results:

no positive control tested

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

Any other information on results incl. tables

SIGNS OF IRRITATION DURING INDUCTION:
- Slight to moderate (Grade 1 and 2) reactions in test group

- Slight irritation in control group

EVIDENCE OF SENSITIZATION OF EACH CHALLENGE CONCENTRATION:
- None

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not sensitizing when applied to guinea pigs.

Executive summary:

The study was performed 1986 as GLP-test following EC-test method B.6 as Maximization test (Magnusson & Kligman) on female guinea pigs. There i s no evidence from the test results that the test material is a sensitiser in guinea pigs after induction at a concentration of 10% (injection and topical ) and challenge at a concentration of 25%. Vehicle was paraffin oil. In conclusion, the test item is not sensitizing.