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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The test was conducted in compliance with OECD Guideline for Testing Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay". Adopted 24 April 2002. There was no deviation from the protocol.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
m-BP
IUPAC Name:
m-BP
Constituent 2
Chemical structure
Reference substance name:
3-{[4'-(3-aminophenoxy)-[1,1'-biphenyl]-4-yl]oxy}aniline
EC Number:
700-102-1
Cas Number:
105112-76-3
Molecular formula:
C24H20N2O2
IUPAC Name:
3-{[4'-(3-aminophenoxy)-[1,1'-biphenyl]-4-yl]oxy}aniline
Details on test material:
- Name of test material (as cited in study report): 3,3-(4,4-diphenylenedioxy)dianiline
- Substance type: Raw material for polymer
- Physical state: Off-white powder
- Analytical purity: >99 %
- Lot/batch No.: 20070501
- Expiration date of the lot/batch: End April 2008
- Storage condition of test material: Room temperature
- Other: Date of receipt was 28 August 2007

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Ltd., Bicester, Oxon, England
- Age at study initiation: twelve weeks
- Weight at study initiation: 19.1 to 23 g
- Housing: animals were housed inside a barriered rodent facility (Building F21, Room 12). They were housed individually in polycarbonate cages with woodflake bedding. The mice were given Nestlets for environmental enrichment.
- Diet (e.g. ad libitum): free access to standard rodent diet (RM1(E) SQC expanded pellet)
- Water (e.g. ad libitum): potable water from the public supply was freely available via polycarbonate bottles fitted with sipper tubes
- Acclimation period:six days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25 and 50 % w/v
No. of animals per dose:
Four
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
- Irritation:
- Lymph node proliferation response:


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response:


TREATMENT PREPARATION AND ADMINISTRATION:
The maximum practical concentration for pinna dosing was 50 % w/v in DMF. Based on this information the following concentrations were selected for the study:
10, 25 and 50 % w/v

The dosage for the positive control group was chosen based on previous study conducted at this laboratory using HCA in DMF and was 25 % v/v.

Groups of four mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 μl of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner, and a further group of four mice received HCA (positive control) in the same manner.

In the main study, five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline containing 3H-methyl Thymidine† (3HTdR: 80 μCi/ml) giving a nominal 20 μCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle (26 gauge) after the mouse had been heated in a warming chamber.
† Specific activity 2.0 Ci/mmol, concentration 1.0 mCi/mL

All animals were observed daily for signs of ill health or toxicity. The ears were also examined for signs of irritation.

In the main study, five hours following the administration of 3HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised and pooled for each experimental group. 1.0 ml of phosphate buffered saline was added to the pooled lymph nodes for each group. The animals were then discarded and no further investigations were carried out. The following procedures were carried out with the lymph nodes from these animals only.

A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The pooled LNC were then washed by adding 10 ml phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 ml trichloroacetic acid (TCA: 5%) following the final wash.

After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 ml 5% TCA and transferred to 10 ml Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by β-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes
relative to that recorded for control nodes (test/control ratio).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The stimulation index for the positive control substance hexyl cinnamic aldehyde (HCA), was 5.5 which proved to demonstrate the reliability and sensitivity of this assay to detect skin sensitization potential in this laboratory.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The stimulation indices were obtained by comparing the proliferation in the vehicle (DMF) treated group with the values from the three test groups. These indices were 0.7, 0.6 and 0.6 for the three test groups. The stimulation for the postive control substance HCA was 5.5.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
The DPM in the control group (DMF only) showed a dpm of 2256 (282 dpm per node). The test group with a concentration of 10 % w/v of the test substance in DMF showed a dpm of 1657 (207 dpm per node). The test group with a concentration of 25 % w/v showed a dpm of 1288 (161 dpm per node). The test group with a concentration of 50 % w/v showed a dpm of 1348 (168.5 dpm per node).The positive control group with a concentration of 25 % v/v hexyl cinnamic aldehyde had a dpm of 12359 (1545 dpm per node).

Any other information on results incl. tables

Table 1: Group dpm/node and test/control ratios (stimulation indices)

Group

Concentration in vehicle % w/v

Dpm

Number of lymph nodes per group

Dpm/node

Test/control ratio

Result*

1

DMF

2255.85

8.0

281.98

n.a.

n.a.

2

10

1656.95

8.0

207.12

0.7

Negative

3

25

1288.25

8.0

161.03

0.6

Negative

4

50

1348.25

8.0

168.53

0.6

Negative

5

HCA 25 % v/v

12359.1

8.0

1544.88

5.5

Positive

* Test/control ratios of 3 or greater indicate a positive result

n.a.: not applicable

dpm: Disintegrations per minute (less background count of 71.35 dpm)

DMF: Dimethylformamide (vehicle control)

HCA: Hexyl cinnamic aldehyde (positive control)

Table 2: Group dpm/node and test/control ratios (stimulation indices)

Group

Concentration in vehicle % w/v

Dpm

Number of lymph nodes per group

Dpm/node

Test/control ratio

Result*

1

DMF

2255.85

8.0

281.98

n.a.

n.a.

2

10

1656.95

8.0

207.12

0.7

Negative

3

25

1288.25

8.0

161.03

0.6

Negative

4

50

1348.25

8.0

168.53

0.6

Negative

5

HCA 25 % v/v

12359.1

8.0

1544.88

5.5

Positive

* Test/control ratios of 3 or greater indicate a positive result

n.a.: not applicable

dpm: Disintegrations per minute (less background count of 71.35 dpm)

DMF: Dimethylformamide (vehicle control)

HCA: Hexyl cinnamic aldehyde (positive control)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
3,3-(4,4-biphenylenedioxy)dianiline is not regarded as a potential skin sensitiser.
Executive summary:

The skin sensitisation potential of 3,3 -(4,4 -biphenylenedioxy)dianiline using the murine local lymph node assay (LLNA) was assessed in compliance with OECD Guideline for Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay".

In each phase of the study, the female CBA mice were treated by daily application of 25 L of each of one of these concentrations (10, 25, or 50% w/v), or control (vehicle or positive), to the dorsal surface of both ears for three consecutive days. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assess five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine by beta-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio - stimulation index). The test substance is regarded as a sensitizer if at least once concentration of the chemical results in a three-fold grater increase in 3HTdR incorporation compared to control values. In this assay the test/control ratios obtained for 10, 25 and 50% w/v were 0.7, 0.6, 0.6 respectively which indicates that 3,3 -(4,4 -biphenylenedioxy)dianiline did not show the potential to induce skin sensitization. The test substance therefore is not regarded as a potential skin sensitizer.