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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 June 2007 to 3 July 2007
Reliability:
1 (reliable without restriction)
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. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

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
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Cellcore MX
- Physical state: solid
- Appearance: black powder
- 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
- Strain: 16 CBA/Ca (CBA/CaBkl) mice; 1 CBA/Ca (CBA/Ca CruBR) mouse
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 15 - 23 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum Certified Rat and Mouse Diet
- Water: ad libitum access to mains tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 – 25 °C
- Humidity (%): 30 – 70 % (relative)
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light (06:00 to 18:00)

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
- Preliminary Screening Test
50 % w/w in vehicle

- Main Test
0, 10, 25 and 50 % w/w in vehicle
No. of animals per dose:
- Preliminary Screening Test
1 female

- Main Test
4 females per dose
Details on study design:
PRELIMINARY SCREENING TEST
A single mouse was treated by daily application of 25 µL of test material at a concentration of 50 % w/w in vehicle, to the dorsal surface of each ear for three consecutive days (days 1, 2 and 3). The mouse was 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. Body weight measurements were taken on day 1 (prior to dosing) and on day 6.
Based on findings from the preliminary screening test, the dose levels selected for the main test were 10, 25 and 50 % w/w in vehicle.

MAIN STUDY
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 and 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 4 animals received the vehicle alone in the same manner.

³H-Methyl Thymidine Administration
Five days following the first topical application of the test material (day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing ³H-methyl thymidine (³HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

OBSERVATIONS
- Clinical observations: all animals were observed twice daily on days 1, 2 and 3 and once daily on days 4, 5 and 6. Any signs of toxicity or signs of ill health were recorded.
- Body weights: body weight measurements were recorded on day 1 (prior to dosing) and on day 6 (prior to termination).

TERMINAL PROCEDURES
5 hours following administration of ³HTdR all mice were sacrificed by carbon dioxide asphyxiation. The draining of the auricular lymph nodes from the four mice of each group were excised and pooled and 1 mL of PBS added.
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 through the gauze with 4 mL of PBS into a petri dish. 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 x gravity) for ten minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5 % trichloroacetic acid (TCA).
After incubation for approximately 18 hours at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 x gravity) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase Trisafe). ³HTdR incorporation was measured by β-scintillation counting. The Poly Q vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes to reduce the risk of luminescence. After 20 minutes the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measure using the Beckman LS6500 scintillation system.

INTERPRETATION OF RESULTS
The proliferation of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ration of ³HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
A test material will be regarded as a skin sensitiser if at least one concentration of the test material results in a threefold or greater increase in ³HTdR incorporation compared to controls.
Positive control substance(s):
other: phenylacetaldehyde (90 %)

Results and discussion

Positive control results:
A study was performed to assess the sensitivity of the strain of mouse used at the test laboratory to a known sensitiser. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 429 and EU Method B.42.
During the study, 3 groups of 5 animals were treated with 50 µL (25 µL per ear) of2-phenylacetaldehyde (90 %) as a solution in propylene glycol at concentrations of 5, 10 and 25 % v/v. A further group was treated with vehicle alone.
The stimulation index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control groups was 11.25, 20.00 and 29.49 at positive control concentrations of 5, 10 and 25 % v/v, respectively. Phenylacetaldehyde (90 %) was therefore confirmed to be a skin sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: A stimulation index of less than 3 was recorded for the three concentrations of test material that were tested (see Table 1).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 3846.51, 5092.86, 4793.05 and 5104.41 disintegrations per minute were recorded for the vehicle and test material concentrations of 10, 25 and 50 % w/w in vehicle, respectively (see Table 1).

Any other information on results incl. tables

Preliminary Screening Test

No signs of toxicity were noted. Black staining on the ears and fur was noted post-dose on days 1, 2 and 3.

Main Test

There were no deaths. No sign of systemic toxicity were noted in the test or control animals during the test. Black staining on the ears and fur was noted in animals treated with the test material at a concentration of 50 % w/w in propylene glycol post-dose on days 1, 2 and 3, and in all test animals on day 4. Body weight 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.

Table 1: Results from the Main Test

Conc. (% w/w) in vehicle

dpm

dpm/node*

Stimulation Index#

Result

0 (vehicle control)

3846.51

480.81

na

na

10

5092.86

636.61

1.32

Negative

25

4793.05

599.13

1.25

Negative

50

5104.41

638.05

1.33

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

Applicant's summary and conclusion

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

The skin sensitisation potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 429 and EU Method B.42.

Following a preliminary screening test, three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test material as a suspension in propylene glycol at concentrations of 10, 25 and 50 % w/w. A further group of four animals was treated with propylene glycol alone.

The stimulation index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was 1.32, 1.25 and 1.33 for test material concentrations of 10, 25 and 50 % w/w in vehicle, respectively.

The test material was therefore considered to be a non-sensitiser under the conditions of the test.