1-(2,4,6-trichlorophenyl)acetone O-methyloxime

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-12-05 until 2013-12-10

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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

2015

Report date:

2015

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

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:

1-(2,4,6-trichlorophenyl)acetone O-methyloxime

EC Number:

807-534-0

Cas Number:

1228284-89-6

Molecular formula:

C10H10Cl3NO

IUPAC Name:

1-(2,4,6-trichlorophenyl)acetone O-methyloxime

Test material form:

other: liquid

Details on test material:

- Physical state: Brown liquid
- Purity test date: 25 September 2013
- Expiration date of the lot/batch: End September 2015
- Stability under test conditions: Stable until 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 mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: ELEVAGE JANVIER Route des Chènes Secs B.P. 4105 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: 8 Weeks
- Weight at study initiation: 21.1-21.9 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. Ssniff Spezialdiäten GmbH, D-59494, Soest, Germany
- Water: Tap water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1-24.5
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 December 2013 To: 10 December 2013 (Preliminary experiment 13 November 2013 to 02 December 2013).

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0 (control), 25, 10, 5%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Vehicle used: Acetone/olive oil (4:1 v/v)
- Compound solubility: Test item was soluble in the vehicle.
- Method: Preliminary Irritation/Toxicity Tests were performed.
Test 1: Test item concentrations of 100% (undiluted) and 50% (w/v) in acetone/olive oil were tested on CBA/J Rj mice (2 animals/dose). The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed.
- Results: No mortality was observed. Marked body weight loss (11.8%) was observed for one animal in the 50% (w/v) dose group. Increased activity / hyperactivity and tremors were observed in the animals in the 100% (undiluted) and 50% (w/v) dose groups on Days 1-3. Scratching was observed in both dose groups on Day 2. Scales was observed in both dose groups on Day 6, furthermore crust at the head was observed for one animal in the 100% (undiluted) dose group on Day 6. Erythema (E=1) was also observed in the animals of the 100% (undiluted) dose group on Day 3. Ear thickness of the animals was measured by using a thickness gauge on Days 1, 3 and 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. Increased ear thickness data were observed in the 100% (undiluted) dose group. The obtained values exceeded the criteria (≥25% increase in ear thickness). The revealing ear punch weights were within the historical control range. The draining auricular lymph nodes of the animals were visually examined: the appearance of the lymph nodes was larger than normal in all animals. Based on these results, the 100% and 50% (w/v) dose groups were not used in the main test and a second preliminary test was undertaken.
Test 2: Test item concentrations of 25% and 10% (w/v) in acetone/olive oil were tested on 2 animals/dose. This test was conducted using the same experimental conditions as the first preliminary experiment.
_ Results: No mortality or signs of systemic toxicity were observed. No marked body weight loss was observed in the 25 and 10% (w/v) dose groups. No significant increase in the ear thickness was detected, and the ear punch weights were within the historical control range. The draining auricular lymph nodes of the animals were visually examined: the appearance of the lymph nodes was larger than normal in the 25% (w/v) dose group and normal in the 10% (w/v) dose group. Based on these results, 25 and 10% (w/v) doses were considered to be acceptable for the main test.

MAIN STUDY
STUDY CRITERIA
- Name of test method: LLNA
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
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: A study was performed to assess the skin sensitization potential of the test item in the CBA/J Rj strain mouse following topical application to the dorsal surface of the ear on days 1, 2 and 3. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 25%, 10% or 5% w/v. The test item formulation was administered using a pipette. A further group of four animals was treated with acetone/olive oil 4:1 alone.

Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing approximately 20 μCi of 3HTdR. Once injected, the mice were left for 5 hours (± 30 minutes).

OBSERVATIONS: All animals were observed at least once daily on Days 1-6. Any signs of toxicity, local irritation or signs of ill health during the test were recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to 3HTdR injection).

TERMINAL PROCEDURES: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes were excised and processed. A single cell suspension of the lymph node cells for each individual animal was prepared and 3HTdR incorporation was determined.

Positive control substance(s):

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

Statistics:

The proliferative response of lymph nodes cells from the lymph nodes of each individual animal was expressed as radioactive disintegrations per minute (DPM) per animal. The average of the two measured DPM values of 5% (w/v) TCA solution was used as background DPM value. The results were expressed as disintegrations per node (DPN = DPM divided by the number of lymph nodes) for each animal following the industry standard for data presentation.

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

The use of the individual approach to calculate SI made the use of a statistical analysis available. The statistical analysis was performed using the SPSS/PC+ (4.0.1) software package. The 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 by CiToxLAB Hungary Ltd. with 25% (w/v) α-Hexylcinnamaldehyde (experimental dates: 27 November 2013 and 04 December 2013). A significant lympho-proliferative response (stimulation index value of 18.5 (study 13/106-037E) and 15.4 (study 13/105-037E), respectively) was noted for α-Hexylcinnamaldehyde. These results demonstrated the appropriate performance of the assay. Furthermore, the mean DPN values observed for the vehicle control in this experiment were within the historical control range.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 1.0, 4.8, 2.1 and 1.2 for 0, 25, 10 and 5% test item in acetone/olive oil 4:1 treated groups, respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Measured DPM/group 171.3, 819.8, 361.8 and 201.3 for 0, 25, 10 and 5% test item in acetone/olive oil 4:1, respectively

Any other information on results incl. tables

No mortality or signs of systemic toxicity were observed during the study. There were no indications of any 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. Enlarged or slightly enlarged lymph nodes were observed in the 25% (w/v) dose group; slightly enlarged lymph nodes were observed in the 10% (w/v) dose group. Normal sized lymph nodes were detected in the 5% (w/v) dose group except for one animal which had slightly enlarged lymph nodes. The observed stimulation index values were 4.8, 2.1 and 1.2 at concentrations of 25, 10 and 5% (w/v), respectively. The calculated EC3 value is 15.0% (w/v).

Table1: DPM, DPN and Stimulation Index Values

Concentration of test substance (%w/v)	Measured DPM	DPM	Number of lymph nodes	DPN	Group DPN	Stimulation Index
0 (vehicle only)	876	841	2	420.5	171.3	1.0
	217	182	2	91.0
	171	136	2	68.0
	246	211	2	105.5
25%	4037	4002	2	2001.0	819.8	4.8*
	1234	1199	2	599.5
	968	933	2	466.5
	459	424	2	212.0
10%	577	542	2	271.0	361.8	2.1
	1320	1285	2	642.5
	327	292	2	146.0
	810	775	2	387.5
5%	314	279	2	139.5	201.3	1.2
	1068	1033	2	516.5
	200	165	2	82.5
	168	133	2	66.5

* = Significant (p<0.05, Mann-Whitney U-test versus negative control)

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, under the conditions of the present assay, the test item tested in a suitable vehicle was shown to have skin sensitisation potential in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of the test substance following dermal exposure to female CBA/J Rj mice.

Following a preliminary screening test, 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). Three groups of 4 mice received 25, 10 and 5% (w/v) test item (formulated in acetone:olive oil 4:1 (v:v) - the negative (vehicle) control group received the vehicle. There was no treatment on Days 4, 5 and 6. 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).

No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. No treatment related effects were observed on body weight. The appearance of the lymph nodes was normal in the negative control group. Enlarged or slightly enlarged lymph nodes were observed in the 25% (w/v) dose group; slightly enlarged lymph nodes were observed in the 10% (w/v) dose group. Normal sized lymph nodes were detected in the 5% (w/v) dose group except for one animal which had slightly enlarged lymph nodes. The observed stimulation index values were 4.8, 2.1 and 1.2 at concentrations of 25, 10 and 5% (w/v), respectively. The calculated EC3 value is 15.0% (w/v).

In conclusion, under the conditions of the present assay, the test item tested in a suitable vehicle was shown to have skin sensitisation potential in the Local Lymph Node Assay.