Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 26 April 2005 and 12 May 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
β,4-dimethylcyclohex-3-ene-1-propan-1-al
EC Number:
229-846-0
EC Name:
β,4-dimethylcyclohex-3-ene-1-propan-1-al
Cas Number:
6784-13-0
Molecular formula:
C11H18O
IUPAC Name:
3-(4-methylcyclohex-3-en-1-yl)butanal
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaBk1) strain mice were supplied by B & K Universal Ltd, Hull, UK and (CBA/Ca CruBR) strain mice were supplied by Charles River UK Limited, Margate, Kent, UK.
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: 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.

- Age at study initiation: eight to twelve weeks old
- 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
- Water: ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25°C
- Humidity: 30 to 70%
- Air changes (per hr): approximately fifteen changes 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.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
This vehicle was chosen as it produced the most suitable formulation at the required concentration.
Concentration:
100%, 50%, 20%
No. of animals per dose:
Five
Details on study design:
PRE-SCREEN TEST: Using all available information 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.
MAIN TEST:
- Test material administration: Groups of five mice were treated with the undiluted test material or the test material at concentrations of 25% or 50% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test material 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 conc entration 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 ov er 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) cont aining 3H-methyl thymidine (3HTdR:80µCi/mL, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total of 20 μCi to each mouse.
- 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. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
-Terminal procedures: 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 indi vidual animal 1 ml of PBS was added to the lymph nodes.
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 r insed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. 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 ce ntrifuge 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 mate rial, the pellet was resuspended in 3 mL of 5% Trichloroacetic acid (TCA).
After approximately 18 hours incubation at approximately 4°C, 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 scin tillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by ß-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample chang er 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).
- Criteria used to consider a positive response: The Proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each in dividual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index (SI)). 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)
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.
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:
Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were 2.76, 3.34 and 8.91 for 5%, 10% and 20% respectively.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
< 25
Key result
Parameter:
SI
Value:
3.73
Test group / Remarks:
25% in 4:1 acetone:olive oil
Key result
Parameter:
SI
Value:
4.18
Test group / Remarks:
50% in 4:1 acetone:olive oil
Key result
Parameter:
SI
Value:
8.26
Test group / Remarks:
100%
Cellular proliferation data / Observations:
Preliminary Screening Test : No signs of systemic toxicity were noted. Based on this information the dose levels selected for the main test were 25% and 50% v/v in acetone/olive oil 4:1 and 100%.
Main Test:
-Estimation of the Proliferative Response of Lymph Node Cells: The radioactive disintegrations per minute (dpm) per lymph nodes for each individual animal and the stimulation index (SI) are given under ‘any other information on results incl. tables’. A stimulation index of greater than 3 was recorded for the three concentrations of the test material (25% and 50% v/v in acetone/olive oil 4:1 and 100%).
-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.

Any other information on results incl. tables









































































































































































Individual Dpm's and Stimulation Index (SI)   
Concentration (% v/v) in acetone/olive oil 4:1 Animal NumberDpml/Animal aMean Dpm/Animal (Standard Deviation)Stimulation Index (SI)bResult
Vehicle1-11282.63 N/AN/A
1-21346.761547.99 (±776.17)
1-31297.13 
1-4912.1 
1-52901.34 
252-14777.33 3.73Positive
2-26964.515769.90** (±1416.21)
2-33806.35 
2-46331.74 
2-56969.55 
503-13772.53 4.18Positive
3-26363.496467.73*** (±1754.85)
3-38666.81 
3-46883.68 
3-56652.14 
1004-113036.53 8.26Positive
4-214905.7412792.82*** (±1609.48)
4-313323.6 
4-412182.08 
4-510516.16 
      
a = Total number of lymph nodes per animal is 2   
b = Stimulation Index of 3.0 or greater indicates a positive result  
N/A = Not applicable     
**  Significantly different from control group p<0.01   
*** Significantly different from control group p<0.001   

Applicant's summary and conclusion

Interpretation of results:
other: Limoxal is classified as a skin sensitiser (1B) in accordance with EU CLP (1272/2008 and its amendments).
Conclusions:
The test material was considered to be a sensitiser under the conditions of the test.
Executive summary:

A study was performed to assess the skin sensitisation potential of limoxal in CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was conducted according to the OECD Guideline 429 and EC Method B42 in compliance with GLP.


Following a preliminary screening test, three groups, each of five animals, were treated with 50 ul (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 25% or 50% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. No signs of systemic toxicity were noted. 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 were 3.73, 4.18 and 8.26 for 25%, 50% and 100% respectively. The EC3 was determined to be below 25%.


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