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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 December 2008 - 27 January 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
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

Constituent 1
Test material form:
other: liquid (not specified)
Details on test material:
Description: dark brown liquid
Storage conditions: room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca (CBA/CaOlaHsd).
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: 15 - 23 g.
- Housing: 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.
- Diet (e.g. ad libitum): ad libitum.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C.
- Humidity (%): 30 - 70 %.
- Air changes (per hr): Approximately 15.
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

IN-LIFE DATES: From: 17 December 2008 To: 27 January 2009

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
Concentration:
10, 25 and 50 % v/v in dimethyl formamide
No. of animals per dose:
4 animals per dose
Details on study design:
PRELIMINARY SCREENING TEST
Using available information regarding the irritancy potential of the test material, a preliminary screening test was performed using three mice, one mouse per test material concentration. The mice were treated by daily application of 25 µL of the test material at concentrations of 10, 25 or 50 % v/v in dimethyl formamide, to the dorsal surface of each ear for 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 TEST
Test Item Administration:
Groups of four mice were treated with the test item at concentrations of 10, 25 or 50 % v/v in dimethyl formamide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 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 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 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:
Approximately 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 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 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).

Determination of 3HTdR Incorporation
After approximately eighteen hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 x gravity) for ten 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. The purpose of this period of time in darkness was to 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.


INTERPRETATION OF RESULTS
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) 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 material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in ³HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in ³HTdR incorporation will be classified as a "non-sensitiser".
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Study dates: 29 October 2008 - 4 November 2008

Methods
One group of five animals was treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde, Tech, 85 % as a solution in dimethyl formamide at a concentration of 15 % v/v. A further control group of five animals was treated with dimethyl formamide alone.

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 Stimulation Index Result
dimthyl formamide
15 4.55 Positive

Conclusion
α-Hexylcinnamaldehyde was considered to be a sensitiser under the conditions of the test

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The stimulation indices are given in Table 1. A stimulation index of greater than 3 was recorded for the test material at concentrations of 10 and 25 % v/v in dimethyl formamide. A stimulation index of less than 3 was recorded for the test material at a concentration of 50 % v/v in dimethyl formamide.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute are given in Table 1.

Any other information on results incl. tables

Preliminary Screening Test

No signs of systemic toxicity were noted.

 

Main Test

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.

 

Table 1 Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% v/v) in dimethyl formamide

dpm

dpm/Node*

Stimulation Index

Result

Vehicle

5241.82

655.23

-

-

10

36365.72

4545.72

6.94

Positive

25

18493.64

2311.71

3.53

Positive

50

13414.80

1676.85

2.56

Negative

*Obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be a sensitiser under the conditions of the test.
Executive summary:

The sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 429 and EU Method B.42.

Female CBA mice were treated with the test material as a solution in dimethyl formamide at concentrations of 10, 25 or 50 % v/v. The Stimulation Indices in the 10 and 25 % v/v dose groups were greater than 3.

As a result, the test material was considered to be a sensitiser under the conditions of the test.

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