4-(trifluoromethoxy)aniline

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 2013 through April 2013

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

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)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-(trifluoromethoxy)aniline

EC Number:

207-317-5

EC Name:

4-(trifluoromethoxy)aniline

Cas Number:

461-82-5

Molecular formula:

C7H6F3NO

IUPAC Name:

4-(trifluoromethoxy)aniline

Test material form:

other: solution

Details on test material:

2 TEST AND REFERENCE ITEMS
2.1 Test Item
Information as provided by the Sponsor.
Identification: 4-(Trifluoromethoxy) Aniline
Batch: KLP006B041209014
Purity: >99%
Stability in solvent: Not indicated by the sponsor
Expiry Date: August 31, 2013
Storage Conditions: (provided by the Sponsor): At room temperature, protected from light and humidity, under Nitrogen

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

3 MATERIALS AND METHODS

3.1 Definitions and Abbreviations
BG Background (1 mL 5% trichloroacetic acid) in duplicate
DPM Radioactive disintegrations per minute
EC3 Estimated Concentration for a S.I. of 3
3HTdR 3H-methyl thymidine
LLNA Local Lymph Node Assay
PBS Phosphate buffered saline
S.I. Stimulation Index
SD Standard Deviation

3.2 Test System

Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system.
Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
Number of animals for the pre-test: 4 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age: 1st and 2nd pre-test: 9 - 10 weeks (beginning of treatment) Main study: 10 - 11 weeks (beginning of treatment)
Body weight: see Appendix 1 and 2
Identification: The animals were distributed into the test groups at random. All animals belonging to the same
Harlan Study Number: 1533100 Report Page 11
experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

5, 10, 25

No. of animals per dose:

4

Details on study design:

3.9 Experimental Design and Procedures

3.9.1 Topical application

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1, v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

3.9.2 Administration of 3H-methyl-thymidine

Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.9 μCi of 3H-methyl thymidine (equivalent to 79.4 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

3.9.3 Determination of incorporated 3HTdR

Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells
Harlan Study Number: 1533100 Report Page 14
were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

3.9.4 Interpretation of raw data

The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that 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.

3.10 Observations

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:

Mortality / Viability: At least once daily from experimental start to necropsy.
Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily.
Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

3.11 Statistical Analysis
The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:

3.12 Positive Control Data
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2012, see Appendix 3 and 4.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

5%

Results 3

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

25%

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see table on page 16 of the attached study report

Any other information on results incl. tables

4 RESULTS AND DISCUSSION

4.1 Results

4.1.1 Calculation and Results of Individual Data

see table on page 16 of the attached study report

4.1.2 Viability / Mortality

No deaths occurred during the study period.

4.1.3 Clinical Signs

No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

4.1.4 Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

The individual body weight values are included in Annex 2.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

4.2 Discussion
In order to study a possible skin sensitising potential of 4-(Trifluoromethoxy) Aniline, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1, v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of four mice was treated with the vehicle (acetone:olive oil (4+1, v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.
All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.
A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices of 0.8, 1.4, and 1.4 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1, v/v)
The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.
5 CONCLUSION
The test item 4-(Trifluoromethoxy) Aniline was not a skin sensitiser under the test conditions of this study.

Executive summary:

SUMMARY

In the study the test item 4-(Trifluoromethoxy) Aniline formulated in acetone:olive oil (4+1, v/v) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 0.8, 1.4, and 1.4 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in acetone:olive oil (4+1, v/v), respectively.

The test item 4-(Trifluoromethoxy) Aniline was not a skin sensitiser under the test conditions of this study.