N-methoxy-1-(2,4,6-trichlorophenyl)propan-2-amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 January 2014 to 14 January 2014 (06 November 2013 to 18 November 2013 preliminary study)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

EC Number:

941-634-6

Cas Number:

1228284-78-3

Molecular formula:

C10H12Cl3NO

IUPAC Name:

methoxy[1-(2,4,6-trichlorophenyl)propan-2-yl]amine

Test material form:

other: liquid

Details on test material:

- Physical state: Brown liquid
- Expiration date of the lot/batch: Recertification date end September 2015
- Storage condition of test material: Room temperature (<30°C), protected from light and humidity

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J Rj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: 19.0-20.2 g
- Housing: Group caging in Type II. polypropylene / polycarbonate cages
- Diet: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" ad libitum
- Water: Tap water ad libitum
- Acclimation period: 6 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1-25.0
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 09 January 2014 To: 14 January 2014 (preliminary experiment 06 November 2013 to 18 November 2013)

Study design: in vivo (LLNA)

Vehicle:

other: acetone:olive oil (4:1 v/v)

Concentration:

0 (vehicle control), 5, 10, 25 % (w/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test using Acetone: Olive oil 4:1 (v/v) mixture (AOO). A formulation at 50% (w/v) was suitable for the test.
- Irritation/toxicity/ear thickness: Two preliminary irritation/toxicity tests were carried out on 2 mice/dose level (test 1: 100%, 50% in AOO; test 2: 25%, 10%, 5% in AOO). Ear thickness was measured using a thickness gauge on Days 1, 3 and 6. Additional quantification of the ear thickness by ear punch weight determination after euthanasia

Based on these results, 25, 10 and 5% (w/v) doses were considered to be acceptable for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Proliferation assay
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- TOPICAL APPLICATION: Each mouse was topically dosed on the dorsal surface of each ear with 25 μL of the appropriate test item applied using a pipette. Each animal was dosed once a day for 3 consecutive days (Days 1, 2 and 3).

- PROLIFERATION ASSAY: On Day 6, each mouse was iv injected via the tail vein with 250 μL of sterile PBS containing approximately 20 μCi of 3HTdR. Five hours (± 30 minutes) after iv injection, the mice were killed by asphyxiation with CO2 and the draining auricular lymph nodes removed. A single cell suspension of lymph node cells was prepared by gentle mechanical disaggregating of the lymph nodes through a cell strainer. The cell strainer was washed with PBS and the lymph node cells for each mouse were pelleted in a centrifuge. After centrifugation supernatants were discarded and pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for the lymph nodes of each individual animal.

After the final washing step, supernatants were removed. Pellets were gently agitated re-suspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight incubation at 2-8 °C, precipitates were centrifuged and supernatants removed. Pellets were re-suspended in 1 mL of 5% (w/v) TCA solution and dispersed using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).

The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

All animals were observed at least once daily for any clinical signs, including local irritation and systemic toxicity. Individual body weights were recorded on Day 1 (beginning of the assay) and Day 6 (prior to 3HTdR injection).

DPM was measured for each animal. The measured DPM values were corrected with the background DPM value (“DPM”). The measured DPM value of 5 % (w/v) TCA solution was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.

Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

Positive control substance(s):

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

Statistics:

Heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the result was positive, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test.

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed with 25% (w/v) α-Hexylcinnamaldehyde within 6 months of the current study (10 December 2013).

No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. A significant lympho-proliferative response (stimulation index value of 13.0) was noted for α-Hexylcinnamaldehyde in this experiment. The results of the positive control group demonstrated the appropriate performance of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Test group / Remarks:

Control

Remarks on result:

other: The observed stimulation index values were 1.0, 7.4, 2.4 and 1.7 at concentrations of 0 (control), 25, 10, and 5% (w/v), respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM were 139.0, 1025.3, 339.1 and 235.3 at concentrations of 0 (control), 25, 10, and 5% (w/v), respectively.

Results 3

Parameter:

SI

Value:

7.4

Test group / Remarks:

High dose (25%)

Results 4

Parameter:

SI

Value:

2.4

Test group / Remarks:

Mid dose (10%)

Results 5

Parameter:

SI

Value:

1.7

Test group / Remarks:

Low dose (5%)

Any other information on results incl. tables

No mortality or signs of systemic toxicity was observed for the test item treated animals during the study. There were no indications of irritancy at the site of application. No treatment related effects were observed on body weight in the test item treated groups. The appearance of the lymph nodes was normal in the negative control group. In the 25% (w/v) test item treated group larger than normal lymph nodes were observed with slightly enlarged lymph nodes noted in the 10 and 5% (w/v) test item treated groups. The observed stimulation index values were 7.4, 2.4, and 1.7 at concentrations of 25, 10, and 5% (w/v), respectively. Based on these data, the calculated EC3 value for this test item was 11.8%.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was shown to have skin sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Executive summary:

The test item solutions were applied on the dorsal surface of ears of female CBA/J Rj mice (25 μL/ear) for 3 consecutive days (Days 1, 2 and 3). On Day 6, 5 hours prior to termination, animals were intravenously injected via the tail vein with tritiated methyl thymidine (3HTdR). Cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the values obtained were used to calculate stimulation indices (SI).

In the main study, sixteen female CBA/J Rj mice were allocated to four groups of four animals each: - three groups received 25, 10, and 5% (w/v) of the test item (formulated in acetone:olive oil (4:1); negative control group received vehicle alone.

No mortality or signs of systemic toxicity was observed for the test item treated animals during the study. There were no indications of irritancy at the site of application. No treatment related effects were observed on body weight in the test item treated groups. The appearance of the lymph nodes was normal in the negative control group. In the 25% (w/v) test item treated group larger than normal lymph nodes were observed with slightly enlarged lymph nodes noted in the 10 and 5% (w/v) test item treated groups. The observed stimulation index values were 7.4, 2.4, and 1.7 at concentrations of 25, 10, and 5% (w/v), respectively. Based on these data, the calculated EC3 value for this test item was 11.8%.

The test item was shown to have skin sensitisation potential (sensitizer) in the Local Lymph Node Assay.