Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 April 2010 to 13 May 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. As the study was performed with dibromomethane, the fully justifiable read-across to this test material means that the study needs to be allocated a reliability score of 2.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
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:
- Name of test material (as cited in study report): Dibromomethane
- Substance type: clear, colourless liquid
- Physical state: liquid
- Analytical purity: minimum 99.5%
- Composition of test material, percentage of components: bromoform maximum 0.5%; bromochloromethane maximum 0.5%; dichloromethane maximum 0.5%; stabiliser maximum 50 ppm
- Lot/batch No.: 720100043
- Expiration date of the lot/batch: not supplied (12.02.2012 according to certificate of analysis)
- Stability under test conditions:
- Storage condition of test material: room temperature, in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: supplied by Harlan Laboratories UK Limited, Bicester, Oxon, UK.
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: 15-23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): free access to 2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK
- Water (e.g. ad libitum): free access to mains tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): within range 19-25 ºC
- Humidity (%): within range 30-70%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12hr continuous light/12 hr continuous darkness

IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25% or 50% v/v in acetone/olive oil 4:1
No. of animals per dose:
Five
Details on study design:
RANGE FINDING TESTS:
A preliminary screening test was performed using three mice, one per test material concentration. The mice were treated by daily application of 25 µl of the undiluted test material or the test material at concentrations of 50% or 25% v/v in acetone/olive oil 4:1 to the dorsal surface of each ear for the three consecutive days (days 1, 2, 3). The mice were observed twice daily on days 1, 2 and 3 and once daily on days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of each mouse was recorded on day 1 (prior to dosing) and on day 6.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
Groups of five mice were treated with undiluted test material or the test material at concentrations of 50% or 25% in acetone/olive oil 4:1. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (days 1, 2, 3). The test material 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 five mice received the vehicle alone in the same manner.

The positive control animals were similarly treated to the test animals except that 25 µl of the positive control material, a-hexylcinnamaldehyde, technical material 85%, at a concentration of 15% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

Five days following the first topical application of the test material, vehicle control or positive control material (day 6) all mice were injected via the tail vein with 250 µl of phosphated buffer saline (PBS) containing 3H-methyl thymidine (3HTdR:80µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20µCi 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 followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 ml of PBS was added to the lymph nodes.

Preparation of cell suspension: a single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all reamining lymph node cells and these were added to the centrifuge tube. The 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 radioactive material, the pellet was resuspended in 3ml of 5% trichloracetic acid (TCA).

Determination of 3HTdR incorporation: After approximately eighteen hours incubation at approximately 4 ºC, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid. 3HTdR incorporation was measured by ß-scintillation counting. The vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was reduce the risk of luminescence, 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 measured using the Beckman LS6500 scintillation system.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations were appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeniety of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets, Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.

Results and discussion

Positive control results:
a-Hexylcinnamaldehyde tech. 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 15% v/v in acetone/olive oil 4:1.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactiev incorporation of the vehicle control group are as follows: Treatment group Concentration Stimulation Index Result Test Material 25% v/v in acetone/olive oil 4:1 0.70 Negative 50% v/v in acetone/olive oil 4:1 0.63 Negative 100% 1.39 Negative Positive Control Material 15% v/v in acetone/olive oil 4:1 4.10 Positive
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table 2 below

Any other information on results incl. tables

Preliminary screening test

Clinical observations, bodyweight and mortality data are given in Table 1. No signs of systemic toxicity were noted. Based on this information the undiluted test material and the test material at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

Table 1: Clinical observations, bodyweight and mortality data - preliminary screening test

Concentration (% v/v) in acetone/ olive oil 4:1 Animal number Bodyweight (g) Day
Day 1 Day 6 1 2 3 4 5 6
Pre-dose Post dose Pre-dose Post dose Pre-dose Post dose
25 S-1 18 18 0 0 0 0 0 0 0 0 0
50 S-2 20 18 0 0 0 0 0 0 0 0 0
100 S-3 22 21 0 0 0 0 0 0 0 0 0

Main test

Estimation of the proliferative response of lymph node cells

The radioactive disintegrations per minute per lymph nodes for each individual animal and the stimulation index are given in Table 2.

Table 2: Individual disintegrations per minute and stimulation indices

Treatment group Animal number dpm/Animala Mean dpm/Animal Stimulation Indexb Result
Vehicle acetone/olive oil 4:1 1-1 1562.78 1851.80 (±510.73) N/A N/A
1-2 1899.39
1-3 1203.46
1-4 2562.57
1-5 2030.82
Test material 25% v/v in acetone/olive oil 4:1 2-1 1554.68 1292.59 (±191.96) 0.70 Negative
2-2 1030.2
2-3 1262.52
2-4 1372.92
2-5 1242.62
Test material 50% v/v in acetone/olive oil 4:1 3-1 1683.37 1164.78 (±393.53) 0.63 Negative
3-2 1200.50
3-3 874.21
3-4 693.55
3-5 1371.96
Test Material 100% 4-1 1664.36 2572.95 (±1048.91) 1.39 Negative
4-2 1237.47
4-3 3402.07
4-4 3053.08
4-5 3507.75
Positive control material 15% v/v in acetone/olive oil 4:1 5-1 10498.00 7585.69 (±3419.78) 4.10 Positive
5-2 11702.14
5-3 6150.33
5-4 3438.14
5-5 6139.84

a Total number of lymph nodes per animal is 2

b Stimulation Index of 3.0 or greater indicates a positive result

Clinical observations and mortality data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight

Bodyweight changes of the test animals between day 1 and day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

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

A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

- OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

- Method B42 Skin Sensitisation (Local Lymoh Node Assay) of Commission Regulation (EC) No. 440/2008

- United States Environmental Protection Agency Health Effects Test Guidelines OPPTS 870.2600 Skin Sensitisation March 2003

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of the undiluted test material or the test material as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of five animals were treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, a-Hexylcinnamaldehyde tech., 85%, at a concentration of 15% v/v in acetone/olive oil 4:1.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Treatment group Concentration Stimulation Index Result
Test Material 25% v/v in acetone/olive oil 4:1 0.7 Negative
50% v/v in acetone/olive oil 4:1 0.63 Negative
100% 1.39 Negative
Positive Control Material 15% v/v in acetone/olive oil 4:1 4.1 Positive

The test material was considered to be a non-sensitiser under the conditions of the test. a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 15% v/v in acetone/olive oil 4:1.