Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 January 2011 and 22 February 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was conducted in accordance with OECD Guideline 429 "Skin Sensitisation: Local Lymph Node Assay". This study was also performed in accordance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: 3-trifluoromethylaniline
Description: Extremely Pale Yellow Liquid
Batch Number: 101009
Purity: 99.66 %
Date Received: 25 October 2010
Storage Conditions: Room temperature in the Dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd, Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nuliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.

The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014 Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.

The temperature and relative humidity were controlled to remain within target ranges of 19 to 25 degrees and 30 to 70 % respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

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.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50 %, 25 % and 10 %
No. of animals per dose:
4 animals per dose.
Details on study design:
TEST ITEM ADMINISTRATION
The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at a concentration of 50 % (v/v) in acetone/olive oil 4:1. The mice were treated by daily application of 25 uL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test item 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 in the same manner.

3H-METHYL THYMIDINE ADMINISTRATION
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 uL of phosphate buffer saline (PBS) containing 3H-methyl thymidine (3HTdR: 8uCi/mL, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 uCi to each mouse.

OBSERVATIONS
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 test were recorded.

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

TERMINAL PROCEDURES
Termination: Five hours following the 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 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 trough 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 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 precipitate out the readioactive item, the pellet was resuspended in 3 mL of 5 % Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4 degrees celsius, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase "Trisafe"). 3HTdR incorporation was measured by beta-scintillation counting. The Poly Q vials containing the samples and scintillation fluid were placed int he sample changer of the scintillator and left for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of lumiescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measures using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The stimulation index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:
Concentration (% v/v) in acetone/olive oil 4:1: 25
Stimulation Index: 7.25
Result: Positive

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Refer to Table 1.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Refer to Table 1.

Any other information on results incl. tables

Table 1. Disintegrations per minute disintegrations per minute/node and stimulation index

 Concentration (% v/v) in acetone/olive oil 4:1  DPM  DPM/Node *  Stimulation Index **  Result
 Vehicle  10,609.10  1,326.14  NA  NA
 10  4,385.87  548.23  0.41  Negative
 25  5,530.79  691.35  0.52  Negative
 50  8,419.83  1,052.48  0.79  Negative

DPM = Disintegrations per minute

* = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

** = Stimulation index of 3.0 or greater indicates a positive result

NA = Not applicable

All other raw data can be found in the study report.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test.