Benzenamine, N-phenyl-, styrenated

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-04-17 - 2001-05-18 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

EEC Methods for the determination of toxicity, Annex to Directive 96/54/EC (Official Journal No. L248, 30.9.96), Part B, Method B.6. Skin sensitization

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 406 "Skin Sensitization". Adopted 17 July 1992.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

EPA Health Effects Test Guidelines OPPTS 870.2600 "Skin Sensitization" EPA 712-C-98-197. August 1998.

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The present study was conducted for another purpose than REACH prior to the acceptance of OECD or comparable guidelines for a LLNA or in vitro methods.

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, N-phenyl-, styrenated

EC Number:

270-485-3

EC Name:

Benzenamine, N-phenyl-, styrenated

Cas Number:

68442-68-2

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

4-(1-phenylethyl)-N-[4-(1-phenylethyl)phenyl]aniline

Test material form:

liquid: viscous

Remarks:

amber viscous liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: four to seven weeks of age on arrival
- Weight at study initiation: 315 - 450 g at the start of the study (Day 1)
- Housing: in groups of five in suspended metal cages with wire mesh floors. For environmental enrichment, autoclaved hay was given to the guinea-pigs three times weekly at irregular intervals and plastic tubular pipes were included in the cage. These procedures, which alleviate boredom and stereotype behaviours are standard practice at this laboratory and are not considered to have any influence on test results interpretation.
- Diet (e.g. ad libitum): vitamin C enriched guinea-pig diet (Harlan Teklad 9600 FD2 SQC) ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: none stated

ENVIRONMENTAL CONDITIONS
Animal room environmental controls were set to maintain:
- Temperature (°C): 21 ± 3°C
- Humidity (%): 30-70%
Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. These environmental parameters were continuously recorded and the permanent record archived with other departmental raw data.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by means of a time switch to give 12 hours of artificial light (0600 - 1800 hours GMT) in each 24 hour period.
- IN-LIFE DATES: From: To:

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: Alembicol D

Concentration / amount:

0.5% v/v

Day(s)/duration:

on Day 1

Adequacy of induction:

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

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

a 2 x 4 cm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml

Day(s)/duration:

on day 7 for 48 hours

Adequacy of induction:

highest technically applicable concentration used

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: undiluted and in Alembicol D

Concentration / amount:

undiluted and 50% v/v
A 2 x 2 cm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml.

Day(s)/duration:

on day 22, two weeks after second induction, for 24h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 test animals, 5 control animals

Details on study design:

RANGE FINDING TESTS:
TEST SUBSTANCE PREPARATION
A vehicle trial conducted with compound showed that it formed an amber coloured thick liquid at a concentration of 75% v/v in Alembicol D (a product of coconut oil, supplied by Alembic Products, Saltney, Chester, England). The maximum practical concentration was as supplied for topical application and the formulation passed through a needle at 10% v/v (as required for intradermal injections).
The test substance was prepared prior to each application on the day of dosing in Alembicol D. The concentrations used are described in the treatment procedure.
The absorption of the test substance was not determined.
The homogeneity, stability and purity of the test substance were the responsibility of the Sponsor.

TREATMENT PROCEDURE
Preliminary study
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) the minimum irritant test substance concentrations suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration and a dilution of this for the challenge phase.
The animals for the topical irritancy investigations were pre-treated with an intradermal injection of Freund's Complete Adjuvant, 50:50 with water for irrigation (Ph.Eur.), approximately one week prior to the start of the preliminary investigations.
The procedure employed for these investigations was as follows:
Intradermal injections - Intradermal injections (0.1 ml/site) were made into the clipped and shaved flank of two guinea-pigs, using a range of concentrations (0.1 to 10% v/v) of the test item in Alembicol D. The resulting dermal responses were assessed approximately 24 and 72 hours later.
Topical application - Patches of Whatman No. 3 paper (2 cm x 2 cm) were saturated (volume approximately 0.2 ml per patch) with a range of concentrations (25% v/v to as supplied) of the test item in Alembicol D and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of "Blenderm" and firmly secured by "Elastoplast" wound round the trunk and fixed with an impervious plastic adhesive tape. The dressings were removed after an exposure period of approximately 24 hours and the reaction sites were assessed for erythema and oedema (reported as 0 hours). Further examination of the sites was carried out approximately 24 and 48 hours after removal of the dressings.
Selection of concentrations of test substance for the main study
Based on the results of the preliminary investigations, the following concentrations of the test item were selected:
Induction intradermal injection - 0.5% v/v in Alembicol D
This was the highest concentration that caused irritation but did not cause necrosis or give signs of toxicity.
Induction topical application - As supplied
Topical challenge - As supplied and 50% v/v in Alembicol D
From preliminary investigations the test material administered topically as supplied did not give rise to irritating effects.

MAIN STUDY
A. INDUCTION EXPOSURE
Induction intradermal injections - test animals
A 4 x 6 cm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. On Day 1, three pairs of intradermal injections (0.1 ml/site) were made into a 2 x 4 cm area within the clipped area.
Injectables for the test animals were prepared as follows:
1. Freund's Complete Adjuvant was diluted with an equal volume of water for irrigation (Ph.Eur.).
2. Test item, 0.5% v/v in Alembicol D.
3. Test item, 0.5% v/v in a 50 : 50 mixture of Freund's Complete Adjuvant and Alembicol D.
Induction topical application - test animals
The preliminary investigations indicated the test substance applied topically as supplied did not produce skin irritation. Therefore, on Day 7 the same 4 x 6 cm interscapular area was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.5 ml per site of 10% w/w sodium lauryl sulphate in petrolatum. On Day 85 a 2 x 4 cm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml of the test item, as supplied. The patch was placed over the injection sites on the skin of the interscapular region of the test animals and covered by a length of impermeable plastic adhesive tape (5 cm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage (5 cm width "Elastoplast") wound round the torso of the animal and fixed with an impervious plastic adhesive tape. The dressing was left in place for approximately 48 hours.
Induction - control animals
During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.
The dermal reactions for test and control animals to the intradermal injections were recorded 24 hours following the injections and the reactions to the induction topical application were recorded on removal of the bandages.

B. CHALLENGE EXPOSURE
Challenge - control and test animals
The control and test animals were challenged topically two weeks after the topical induction application (Day 22) using the test item, as supplied and 50% v/v in Alembicol D.
Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 2 x 2 cm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of the test item, as supplied and applied to an anterior site on the flank. The test item, 50% v/v in Alembicol D was applied in a similar manner to the posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" (5 cm width) secured with "Elastoplast" (7.5 cm width) wound round the trunk and fixed with an impervious plastic adhesive tape.
The challenge sites were evaluated approximately 24 and 48 hours after removal of the patches.

Positive control substance(s):

yes

Remarks:

The sensitivity of the guinea-pig strain used is checked periodically at the laboratory with hexyl cinnamic aldehyde (HCA), a known moderate sensitizer.

Results and discussion

Positive control results:

RESULTS
INDUCTION
Intradermal injections: Necrosis was recorded at all sites receiving Freund's Complete Adjuvant. Slight irritation was seen in test animals at sites receiving HCA, 10% v/v in Alembicol D and slight irritation was observed in control animals receiving Alembicol D.
Topical application: Slight to moderate erythema was observed in test animals following topical application with HCA, as supplied. No erythema was seen in the control animals receiving a dry patch.
CHALLENGE
Slight to well-defined dermal reactions were observed for all of the ten test animals compared to no dermal reactions in the control animals, therefore the reactions in the test animals represented hypersensitivity and all ten test animals gave positive sensitization responses.
CONCLUSION
In this study HCA produced evidence of skin sensitisation (delayed contact hypersensitivity) in all of the ten animals, thus confirming the sensitivity of the strain of animals and reliability of the experimental technique.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

as supplied and 50% v/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

There were no dermal reactions seen.

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

as supplied and 50% v/v

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

There were no dermal reactions seen.

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

There were no dermal reactions seen.

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

There were no dermal reactions seen.

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

other: not stated

Group:

positive control

Dose level:

Intradermal injection: 10% v/v in Alembicol D; Topical application: As supplied (neat); Challenge application: As supplied (neat) and 50% v/v in Alembicol D

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Slight to well-defined dermal reactions were observed for all of the ten test animals compared to no dermal reactions in the control animals, therefore the reactions in the test animals represented hypersensitivity.

Remarks on result:

positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 406 (GPMT) on the registered substance itself without deviations. The method is considered to be scientifically reasonable with no deficiencies in documentation. Negative and positive controls gave the appropriate response. Hence, the results can be considered as reliable to assess the skin sensitizing potential of the test item. In this study the test item did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the ten test animals. The test item is not considered to have the potential to cause skin sensitization.
As all of the animals gave negative responses, the test item does not require labelling as skin sensitizer.

Executive summary:

This study was performed to assess the skin sensitization potential of the test item using the guinea-pig. The method followed was that described in:

EEC Methods for the determination of toxicity, Annex to Directive 96/54/EC (Official Journal No. L248, 30.9.96), Part B，Method B.6. Skin sensitization.

OECD Guideline for Testing of Chemicals No. 406 "Skin Sensitization". Adopted 17 July 1992.

EPA Health Effects Test Guidelines OPPTS 870.2600 "Skin Sensitization" EPA 712-C-98-197. August 1998.

MAGNUSSON, B. and KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guinea-pig: Identification of contact allergens, Thomas, C.C" Springfield, Illinois, U.S.A.

The guinea-pigs were dosed by intradermal injection and topical application, as these are the routes of exposure required by the test guidelines and method.

Based on the results of a preliminary study and in compliance with the guidelines, the following dose levels were selected:

Intradermal injection: 0.5% v/v in Alembicol D

Topical application: As supplied

Challenge application: As supplied and 50% v/v in Alembicol D

Ten test and five control guinea-pigs were used in this study.

In this study the test item did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the ten test animals. The test item is not considered to have the potential to cause skin sensitization.

As all of the animals gave negative responses, the test item does not require labelling with the risk phrase R43 "May cause sensitization by skin contact" in accordance with Commission Directive 93/21/EEC. The same applies for labelling as skin sensitizer according to Regulation 1272/2008 and amendments.