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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:
7 February 2005 to 9 March 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

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:
other: opaque liquid
Details on test material:
- Appearance: Blue opaque liquid
- 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:
TEST ANIMALS
- Strain: CBA/Ca (CBA/CaBkl)
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum access to Certified Rat and Mouse Diet
- Water: ad libitum access to mains tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 30 to 70 % (relative)
- Air changes: approximately 15 per hour
- Photoperiod (hrs dark / hrs light): the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES: No data

Study design: in vivo (LLNA)

Vehicle:
other: The test material was administered undiluted and in dimethyl formamide
Concentration:
The test material was administered undiluted and at concentrations of 25 and 50 % v/v in dimethyl formamide.
No. of animals per dose:
5 females per dose level
Details on study design:
PRELIMINARY SCREENING TEST
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test material a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 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. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
No signs of systemic toxicity were noted. Staining of the ears and fur was noted one hour post dosing on Day 1 and for the remainder of the test. Based on this information the dose levels selected for the main test were 25 and 50 % v/v in dimethyl formamide and 100 % (undiluted test material).

MAIN TEST
- Test Material Administration
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.

- 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 study 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. 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 Single 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 10 mL 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 lymph node cells were pelleted at 1400 rpm (approximately 190 g) 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 overnight incubation at 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) 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 (Beckman Instruments Inc., Fullerton, CA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for dpm and standard deviations where appropriate. Individual and group mean dpm values were assessed for dose response relationships by analysis of homogeneity 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:
Three groups of five animals were treated with 50 µL (25 µL per ear) of α-hexylcinnamaldehyde as a solution in acetone/olive oil 4:1 at concentrations of 5, 10 and 25 % v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone.
The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was 1.40, 2.23 and 6.09 for the 5, 10 and 25 % v/v dose groups, respectively. The value of 6.09 is considered to be a positive reaction in this test and therefore 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: The stimulation index (SI) for each dose group is given in Table 1. A stimulation index of less than 3 was recorded for the three concentrations of the test material (25 and 50 % v/v in dimethyl formamide and 100 %).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph nodes for each individual animal are given in Table 1.

Any other information on results incl. tables

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Staining of the ears and fur was noted in all test animals one hour post dosing on Day 1 and for the remainder of the test.

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: Individual dpm’s and the Group Stimulation Indices

Concentration (% v/v) in DMF

Animal Number

dpm/Animal*

Mean dpm / Animal

(± SD)

Stimulation Index

Result

Vehicle

1-1

735.47

792.28 ± 186.54

N/A

N/A

1-2

815.97

1-3

1093.83

1-4

722.99

1-5

593.12

25

2-1

686.35

1117.26 ± 307.51

1.41

Negative

2-2

1326.38

2-3

920.22

2-4

1221.06

2-5

1432.27

50

3-1

1002.21

997.71 ± 257.08

1.26

Negative

3-2

1249.93

3-3

1256.64

3-4

758.99

3-5

720.78

100

4-1

743.98

108.64 ± 305.01

1.36

Negative

4-2

1055.03

4-3

1297.37

4-4

836.74

4-5

1470.10

*Total number of lymph nodes per animal is 2

DMF = dimethyl formamide

SD = standard deviation

Applicant's summary and conclusion

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

A study was performed to assess the skin sensitisation potential of the test material in accordance with the standardised guidelines OECD 429 and EU Method B.42 under GLP conditions.

The assessment was carried out in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Following a preliminary screening test, three groups 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 dimethyl formamide at concentrations of 25 and 50 % v/v. A further group of five animals was treated with dimethyl formamide alone.

The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group was 1.41, 1.26 and 1.36 for the 25, 50 and 100 % dose groups, respectively.

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

Under the conditions of this study, the test material was considered to be a non-sensitiser.