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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:
Experimental Starting Date: 03 July 2015 Experimental Completion Date: 16 July 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study used dose levels of 1, 10 and 100% and increases in the SI were seen at all three dose levels, and the SI just exceeded the threshold of 3 atthe highest dose. However, the dose response was not scientifically plausible and an alternative statistical analysis of the data indicated that the substance is not a sensitiser.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.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.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 10% or 1% v/v in acetone/olive oil 4: 1
No. of animals per dose:
5
Details on study design:
Test ItemFor the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4: 1. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given in the procedure section. The vehicle determination record is included in the attached Appendix 2.The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.Positive Control ItemThe positive control item was freshly prepared as a 25% v/v dilution in acetone/olive oil 4:1.Preliminary Screening TestUsing available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item 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. Local skin irritation was scored daily according to the scale included in the attched Appendix 3. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.Main TestTest Item AdministrationGroups of five mice were treated with the undiluted test item or the test item at concentrations of 10% or 1% v/v in acetone/olive oil 4: 1. 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 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 item, a-Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4: 1 was applied to the dorsal surface of each ear.Local skin irritation was scored as described in the preliminary screening test. The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Mean ear thickness changes were calculated as described in the preliminary screening test.3H-Methyl Thymidine AdministrationFive days following the first topical application of the test item, vehicle control or positive control item (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 activity2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.ObservationsClinical 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.Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).Terminal ProceduresTermination: 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 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 labeled with the study number and dose concentration. 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 lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 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 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by P-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 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 20 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, USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical AnalysisData was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability byanalysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett's multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.Probability values (p) are presented as follows: P<0.001 P<0.01 P<0.05 P>0.05******(not significant) A supplemental statistical analysis supplied by the Sponsor is presented in the attached Appendix 5.

Results and discussion

Positive control results:
a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4: 1 thus demonstrating the sensitivity and reliability of the test system.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2.54
Parameter:
SI
Value:
3.2
Remarks on result:
other:
Remarks:
The highest dose (100%) gave a marginal (borderline) positive result (SI = 3.20) and whilst not conclusive the test item would appear to have some sensitization potential.

Any other information on results incl. tables

Preliminary Screening Test

Clinical observations, body weight and mortality data are given in the attached Table 1 and local skin irritation is given in the attached Table 2. The ear thickness measurements and mean ear thickness changes are given in the attached Table 3.

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the undiluted test item and the test item at concentrations of 10% and 1% v/v in acetone/olive oil 4: 1 were selected for the main test.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per animal and the stimulation index are given in the attached Table 4.

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:

TreatmentGroup

Concentration

Stimulation Index

Result

 

 

 

TestItem

1% v/vin

acetone/oliveoil4:1

2.15

Negative

10% v/vin

acetone/olive oil 4:1

2.27

Negative

100%

3.20

Positive

PositiveControlItem

25% v/vin

acetone/olive oil 4:1

12.36

Positive

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5 and local skin irritation is given in Table 6. The ear thickness measurements and mean ear thickness changes are given in the attached Table 7.

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

Body Weight

Individual body weights and body weight change for test and control animals are given in the attached Table 8.

Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

Calculation of EC3 Value

EC3 = c + [[(3-d)/(b-d)] x (a-c)]

a             100

b            3.20

C            10

d            2.27

EC3 = 10 + [[(3-2.27)/(3.20-2.27)] x (100-10)] = 81

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation   (EC3 value) was calculated to be 81%. However, this result was not adopted because an alternative and more sophisticated statistical analysis of the data provided an overall SI index of 2.54, giving the conclusion that the substance is not a sensitizer.

a = lowest concentration giving stimulation index>3b =  actual stimulation index caused by 'a'

c = highest concentration failing to produce a stimulation index of3d =  actual stimulation index caused by 'c'

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
The substance was concluded not to be a sensitizer.
Conclusions:
A standard preliminary statistical evaluation performed on the data calculated the SI to be 3.2, which is of sufficient magnitude to suggest that the substance becharacterized as a sensitizer. However, a more detailed and appropriate ANOVA of properly transformed data shows that all treatment data are similar. Given these results, it is appropriate to pool the SI data from the all treatments. In so doing the average SI for the substance is 2.54 and therefore, the substance should not be considered a sensitizer.a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4: 1 thus demonstrating the sensitivity and reliability of the test system.
Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

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 item or the test item as a solution in acetone/olive oil 4: 1 at concentrations of 10% or 1% v/v. A further group of five animals was 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 sensitizer, a-Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4: 1

 

Results

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:

TreatmentGroup

Concentration

StimulationIndex

Result

 

 

 

TestItem

1% v/vin

acetone/olive oil 4:1

2.15

Negative

10% v/vin

acetone/olive oil 4:1

2.27

Negative

100%

3.20

Positive

Positive ControlItem

25% v/vin

acetone/olive oil 4:1

12.36

Positive

 

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be  81%.

Conclusion

A standard preliminary statistical evaluation performed on the data calculated the SI to be 3.2, which is of sufficient magnitude to suggest that the substance be characterized as a sensitizer. However, a more detailed and appropriate ANOVA of properly transformed data shows that all treatment data are similar. Given these results, it is appropriate to pool the SI data from the all treatments. In so doing the average SI for the substance is 2.54 and therefore, the substance should not be considered a sensitizer.

 

a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4: 1 thus demonstrating the sensitivity and reliability of the test system.