1,3-Benzenedimethanamine, reaction products with polyethylene glycol mono-Bu ether and TDI

Skin sensitisation

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: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2002

Deviations:

yes

Remarks:

Contrary to OECD 429 of 2002, but in accordance with OECD 429 of 2010, scintillation vials were filled with 10 mL of scintillation fluid for 3H-counting. This did not compromise the validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

of 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): WS400517
- Physical state / appearance: White powder
- Purity of test material: Not applicable, UVCB substance (report states "not made available (UVCB)")
- Expiry date of the batch/lot: 21 January 2011
- Storage condition of test material: Room temperature (ca. 20°C) in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Mouse (healthy females only), strain: CBA/Ca with appropriate range of bodyweight at study start.
- Source: Harlan UK.
- Age at treatment start (1st induction): Eight to twelve weeks.
- Weight at treatment start (1st induction): Minimum 17.3 g, maximum 20.7 g.
- Housing: Individual housing in polycarbonate cages inside a barriered rodent facility.
- Bedding material: Woodflake bedding.
- Cage enrichment: Nestlets and plastic shelter
- Diet (ad libitum): Standard rodent diet (Rat and Mouse No. 1 Maintenance Diet) containing no added antibiotic,
chemotherapeutic or prophylactic agent.
- Water (ad libitum): Tap water
- Acclimation period: At least 7 days before treatment start under laboratory conditions.

Analysis of the batch of diet used and water did not provide evidence of contamination that might have prejudiced the study.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Air changes per hour in the animal room: ca. 15
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
There was no mentioning of any deviations from these ranges, which compromised the integrity or validity of the study.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Main Study:
Induction on Days 1, 2 and 3 at the following concentrations of WS400517 in vehicle (w/v):
0% (vehicle control, 4 females), 10% (4 females), 25% (4 females), 50% (4 females).

A preliminary range-finding test with animals was not performed.

No. of animals per dose:

Main Study:
4 female animals per dose

Details on study design:

Compound Solubility:

A vehicle trial has demonstrated that the maximum practical concentration of WS400517 suitable for use in the LLNA is a 50 % (w/v) solution in the vehicle, dimethylformamide (DMF). At this concentration a pale yellow suspension was formed. In acetone:olive oil (4:1 v/v) a suitable 50 % (w/v) test substance suspension or solution was not achievable.

Treatment Preparation and Administration:

On three consecutive days, groups of 4 female mice were treated by topical application to the entire dorsal surface of both ears with 25 μL/ear/day at the following concentrations (w/v) of test substance in the vehicle:

Group 1 (Vehicle Control): 0%,
Group 2 (Low Dose): 10%,
Group 3 (Mid Dose): 25%,
Group 4 (High Dose): 50%

The test substance preparations were prepared on each day of administration and dosed within 4 hours of preparation.

Observations, Measurements and Endpoints (Pooled treatment group approach):

All animals were checked daily for signs of ill health or toxicity. The ears were also examined daily for signs of irritation. In addition, bodyweights were recorded on Days 1 (prior to treatment) and 6 (three days after the third induction administration). On Day 6, all animals were injected into the tail vein 3H-methyl thymidine diluted in phosphate buffered saline at a nominal dose of 20 µCi per mouse, in order to measure lymphocyte proliferation by radioactive labelling. Five hours afterwards the draining (auricular) lymph nodes were excised and pooled for each experimental group. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed on Day 7. Radioactivity was expressed as the number of radioactive disintegrations per minute (dpm). The ratio of the proliferation (reflected by the magnitude of measured dpm/node) in treated groups to that in the vehicle control group, termed the stimulation index (SI) or test/control ratio, was subsequently calculated for each group.

Criteria Used to Consider a Positive Response:

The test substance is regarded as a sensitizer if at least one concentration of the test substance produces a stimulation index (SI) ≥ 3.

Positive control substance(s):

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

Statistics:

Data were not statistically analysed.

Results and discussion

Positive control results:

A stimulation index (SI) of 7.8 was attained in a contemporaneous positive control assay with the same strain of mice (CBA/Ca) in response to 25% v/v hexyl cinnamic aldehyde in dimethylformamide (DMF), thus demonstrating the reliability and sensitivity of this test system and assay to detect skin sensitization potential in this laboratory.

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

>= 1.4 - <= 1.7

Test group / Remarks:

at 10, 25 and 50% test substance

Any other information on results incl. tables

Mortality / clinical signs:

There were no deaths, no signs of ill health or toxicity and no signs of local irritation over the treated area. Wet fur was noted for all control and test animals post-dose from Day 1 or 2 having resolved completely in all animals by Day 4. Dose residue on the ears was also noted for Groups 3 and 4 post-dose from Day 1, 2 or 3 and was still present at termination in one animal in Group 4.

Body weight: There was no indication of adverse effects on bodyweight attributable to treatment with the test substance.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met