m-phenylenebis(methylamine)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-03-03 to 2004-06-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection 2002-12-04; Date of signature 2003-02-13

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

m-phenylenebis(methylamine)

EC Number:

216-032-5

EC Name:

m-phenylenebis(methylamine)

Cas Number:

1477-55-0

Molecular formula:

C8H12N2

IUPAC Name:

1-[3-(aminomethyl)phenyl]methanamine

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): MXDA
- Physical state: Colourless liquid
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaBkl)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: B & K Universal Ltd, Hull, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet: Free access to Certified Rat and Mouse Diet (Code 5LF2) supplied by BCM IPS Limited, London, UK
- Water: Free access to mains tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 degrees Centigrade
- Humidity: 30-70 %
- Air changes: Approximately 15 per hour
- Photoperiod: Controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Preliminary screening test: Undiluted test material, or test material at a concentration of 10 % or 25 % in dimethyl formamide

Main test: zero %, 2.5 %, 5 % or 10 % test material in dimethyl formamide

No. of animals per dose:

Preliminary screening test: Three

Main test: Four per dose level

Details on study design:

PRELIMINARY SCREENING TEST

As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using three mice. The mice were treated by daily application of 25 μL of the undiluted test material, or test material dilutions of 25 % or 10 % in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5, and 6. The mice treated with the undiluted test material and the test material at a concentration of 25 % were killed for humane reasons due to signs of systemic toxicity. Observations for these animals were performed accordingly. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and of the surviving mouse on Day 6.

MAIN TEST

Groups of four mice were treated with the test material at concentrations of 2.5 %, 5 % or 10 % v/v in dimethyl formamide. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone via the same application method.

3H-METHYL THYMIDINE ADMINISTRATION

Five days following the first topical application of the test material (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, specific activity 2.0 Ci/mmoL, Amershal Biosciences UK Ltd) giving a total of 20 μCi to each mouse.

CLINICAL OBSERVATIONS

All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the study were recorded.

BODYWEIGHTS

The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination)

TERMINATION

Five hours after administration of 3HTdR, all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group, 1 mL of phosphate buffered saline (PBS) was added to the pooled lymph nodes.

PREPARATION OF SINGLE CELL SUSPENSION

A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a 10 mL centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To preceipitate out the radioactive material, the pellet was resuspended in 3 mL of 5 % trichloroacetic acid (TCA).

DETERMINATION OF 3HTdR INCORPORATION

After overnight incubation at 4 degrees Centigrade, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase Trisafe). 3HTdR incorporation was measured by β-scintillation counting. The Poly Q vials containing the samples and scintillation fluid were placed in the sample chamber of the scintillator and left for approximately twenty minutes. The period of time in darkness was used to reduce the risk of luminescence, which has been shown to affect reliabilty of results. After approximately 20 minutes, the vials were shaken vigorously and the number of radioactive disintegrations per minute measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA).

INTERPRETATION OF RESULTS

The test substance was regarded as a sensitiser if at least one concentration resulted in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation was classified as a non-sensitiser.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as a ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Results and discussion

Positive control results:

See Appendix 1 (attached)

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.63

Test group / Remarks:

2.5%

Results 2

Key result

Parameter:

SI

Value:

5.21

Test group / Remarks:

5%

Results 3

Key result

Parameter:

SI

Value:

3.66

Test group / Remarks:

10%

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 2 (attached)

Any other information on results incl. tables

PRELIMINARY SCREENING TEST

Clinical observations, bodyweight and mortality data are given in Table 1 (attached). The animals treated with undiluted test material at a concentration of 25 % v/v in dimethyl formamide were killed for humane reasons on days 1 and 2 respectively due to signs of systemic toxicity including hunched posture and ptosis. There were no signs of systemic toxicity noted in the mouse treated at a concentration of 10 % v/v in dimethyl formamide. Based on this information, the dose levels selected for the main test were 2.5 %, 5 % and 10 % v/v in dimethyl formamide.

MAIN TEST

CLINICAL OBSERVATIONS AND MORTALITY DATA

Individual clinical observations and mortality data for test and control animals are given in Table 3 (attached). There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the study. White residual test material was noted on the fur and ears of animals treated with the test material at concentrations of 5 % and 10 % v/v in dimethyl formamide.

BODYWEIGHT

Individual bodyweights and bodyweight changes for test and control animals are given in Table 4 (attached). Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

The test material was considered to be a sensitiser under the conditions of the test.