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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:
June 2006 to August 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Sponsor batch no: 151105Purity: 99.9%

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Nulliparous, non-pregnant female mice of the CBA/CaCrl strain were obtained from Charles River (UK) Ltd, Margate. All animals were given a clinical inspection for ill health on arrival and a sample was weighed. Husbandry: The animals were group housed during acclimatisation andindividually housed from Day -I in cages that conform with the Code of Practice for the Housing and Care of AnimalsUsed in Scientific Procedures' (Home Office, London, 1989). The animal rooms were designed to permit at least 15 airchanges per hour. The temperature and humidity ranges were 19 to 25°C and 40 to 70% respectively. Recordings ofmaximum and minimum temperature and humidity were made twice daily during the week and once a day on Saturdays andSundays. The rooms were illuminated by fluorescent strip­ lights for twelve hours daily.SQC(E) Rat and Mouse Maintenance Diet No 1, from Special Diets Services Ltd, Witham was freely available to the animalsat all times. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer. Mains water was provided, ad libitum, via cage-mounted waterbottles. The water had been periodically analysed for specific contaminants. Certificates of analysis for contaminant levels ineach batch of diet or in the water supply were consigned to central files at this laboratory.Bedding was provided on a weekly basis to each cage by use ofclean Aspen wood chips (Datesand Ltd, Manchester). The bedding had been analysed for specific contaminants and theresults retained on file at Covance. No contaminants were present in diet, water or bedding atlevels which might interfere with achieving the objective of the study.Environmental enrichment:A number of methods are used at Covance to enrich the environment of the animals as detailed in standard operatingprocedures. Clinical Observations:Treated mice were observed twice daily on Days 1 to 5 andonce on Day 6 for clinical signs of reaction to treatment or forirritation or other changes at the sites of application of the test article. All animals were examined at the beginning and end of theworking day throughout the acclimatisation and study periods to ensure they were in good health.Mice were weighed on Day -1 (the day before dosing) and on Day 6 prior to intravenous administration of 3HTdR.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 10 ,25 and 50% w/v
No. of animals per dose:
5 (five)
Details on study design:
A preliminary screening test was performed with one mouse. The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (50% w/v in DMF) to the dorsal surface of each ear for three consecutive days(Days 1, 2 and 3). The mouse was observed daily for five days from the initiation oftreatment. Any signs of toxicity or irritation during this period were recorded. The body weight was recorded on Day-I. The animal was killed by exposure to a risingconcentration of carbon dioxide at the end of the observation period. MAIN STUDYANIMAL ASSIGNMENT AND TREATMENT-Group Allocation:Mice were arbitrarily selected from the delivery boxes and allocated to the appropriatenumber of cages. The condition of the animals was assessed daily throughout theacclimatisation period of 22 days. A second inspection was performed prior to study commencement to ensure the animals were suitable for the study. Overtly healthyanimals were arbitrarily allocated to the study groups on the day prior to commencement of treatment. A unique number inscribed onto the tail with indelible ink on the day priorto dosing individually identified the mice. A colour-coded card on each cage gaveinformation including study number, animal number and sex. Animals were in a body weight range of 17 to 21 g on Day -1. Based on information fromthe supplier the mice were approximately 10 to 11 weeks old on Day I.TREATMENT PREPARATION AND ADMINISTRATION:Formulation of test article :Formulations were freshly prepared as required, usingdimethylformamide, on Days I, 2 and 3. The formulations were stored at room temperature, in sealed, air-tight containersprior to dosing and were used within two hours of preparation. The formulations were mixed by multiple inversion of the containers prior to administration to ensure homogeneity. Concentrations oftest article were expressed gravirnetrically and in terms oftest article received (without regard to purity or active content).Formulation of Positive control:The positive control material, n-Hexylcinnamaldehyde, was formulated at a concentration of2S% in acetone I olive oil( 4: I v/v). Formulations were freshly prepared as required on Days I, 2 and 3. The formulations were stored at roomtemperature, in sealed, air-tight containers prior to dosing andwere used within two hours of preparation except on Day 2 when the formulation was used 2 hours and 10 minutes afterpreparation. Formulation oftritiated thymidine:Tritiated 3H-methyl thymidine (3HTdR), batch 496, was obtained as a I 000 µCi/mL preparation from Amersham. Biosciences UK Limited. An aliquot of9.2 mL phosphate buffered saline was mixed with 0.8 mL 3HTdR shortly beforeintravenous dosing commenced on Day 6. The final product containedHTdR at 80 µCi/mL.Test Article Administration:Each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse wastreated by direct application of the appropriate test or controlformulation (0.025 mUpinna) dispensed from an automaticmicropipette.Treatment regimenThe five groups of five female mice were subjected to application of the vehicle control, positive control or one of thetest formulations to the outer aspect of both the auditory pinnae of each mouse, once daily on Days I, 2 and 3.On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitateintravenous dosing. Each mouse was transferred to acylindrical restrainer. A plastic syringe and fine gaugehypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 µCi of 3HTdR into a tail veinof each mouse by slow bolus injection. After this treatment, the mice were returned to their cages.Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exposure to a risingconcentration of carbon dioxide. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes. Terminal Procedures:Once death had been confirmed each mouse was placed on abench lined with paper with its ventral aspect uppermost. Amid-line incision from the lower jaw to the sternum was made and the cervical structures were exposed by reflecting the skin.The auricular lymph nodes were located and removed using curved end forceps. Any connective tissue was removed fromthe capsule of the nodes. The auricular lymph nodes of each mouse were placed into individual code-labelled petri dishes containing 5 mL phosphate bulTered saline. Tissue Processing:The lymph nodes collected into each petri dish were cut openand disaggregated by squashing the blade. The resultant liquor Page 14 fragments with a sharp was transferred into code-identifiedconical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor wasadded to the first liquor. At each transfer, debris such as fragments of capsule were retained in the petri dish whereverpossible. After S minutes the pooled liquor was filtered into a secondconical tube by transferring the liquor into a 10 mL syringe and passing it through a 200 µm mesh stainless steel gauzecontaining a fabric filter, cut to size (Clarcor UK, Lockcrtex Filtration Products, Warrington, UK). Any visible sedimentremaining prior to filtering was left in the conical tube. The liquor was centrifuged at 190 G for 10 minutes. Following centrifugation, the supernatant was discarded and the pellet was resuspended in 5 mL phosphate buffered saline. This wascentrifuged at 190 G for IO minutes. The supernatant was discarded and the pellet resuspended in 3 mL of 5% w/vaqueous trichloroacetic acid. The suspension was stored for18 hours at I to 10°C (nominal 4°C). On the following day the suspension was re-centrifuged at190 G for IO minutes and the supernatant was drawn off anddiscarded. The pellet was resuspended in I mL 5% w/v aqueous trichloroacetic acid, then subjected to ultrasonicdispersion for 25 minutes to ensure an homogenoussuspension. The suspension (I mL) was transferred to ascintillation vial and scintillation fluid (ca JO mL) was added.Scintillation Counting:Once prepared the scintillation vials were placed in theappropriate carrier racks. Two background vials were prepared, one containing ca IO mL of scintillation fluid and the othercontaining I mL of 5% w/v aqueous trichloroacetic acid and ca IO mL scintillation fluid. The carrier rack was passed into thescintillation counter. All vials, including the backgroundsamples, were submitted for liquid scintillation counting for IO minutes, using a 'H quench curve.Incorporation of 3HTdR is measured by 8-scintillation countingas disintegrations per minute (DPM) over a ten-minute period. This value was corrected to account for the backgroundcontaining 5% w/v aqueous trichloroacetic acid and scintillation fluid,
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The scintillation counter printed data including the DPM value (disintegrations per minute during a ten minute period) for eachindividual animal. The DPM value was transformed into a mean DPM value for each group. The mean DPM value foreach test group was divided by the mean DPM for the control group to provide the Stimulation Index (SI) value for each test group.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
other: DPM
Value:
194
Variability:
+- 149
Test group / Remarks:
Group 1: Vehicle
Parameter:
other: DPM
Value:
141
Variability:
+-62
Test group / Remarks:
Group 2: 10% w/v
Parameter:
other: DPM
Value:
105
Variability:
+-26
Test group / Remarks:
Group 3 : 25% w/v
Parameter:
other: DPM
Value:
72
Variability:
+-41
Test group / Remarks:
Group 4: 50% w/v
Parameter:
other: DPM
Value:
4 778
Variability:
+-746
Test group / Remarks:
Positive Control
Parameter:
SI
Value:
0.73
Test group / Remarks:
Group 2: 10% w/v
Parameter:
SI
Value:
0.54
Test group / Remarks:
Group 3: 25% w/v
Parameter:
SI
Value:
0.37
Test group / Remarks:
Group 4: 50% w/v
Parameter:
SI
Value:
24.63
Test group / Remarks:
Positive Control

Any other information on results incl. tables

Preliminary screening test

The animal survived and no signs of systemic toxicity or excessive irritation were noted.

Based on this information the dose levels selected for the main test were 10%, 25% and

50% w/v in DMF.

Main test:

Mortality

All animals survived treatment with the test article.

Clincal Signs:

There were no clinical signs indicative of a systemic effect of treatment among mice

treated with the vehicle or with 10, 25 or 50% w/v formulations of the test article.

White residue to the tips of the ears was noted in animals dosed with the test article at a

concentration of 50% w/v in DMF. This was noted on all days except Day 2.

Wet fur around the ears was noted in the positive control animals on Day I. Greasy fur

around the head and redness of the ears was noted from Day 2 to Day 4 with greasy fur

around the head and neck on Days 5 and 6.

Body weights:

There was no indication of a treatment-related effect on body weight.

Stimulation indices

Test results are expressed in terms of Stimulation Indices, the ratios of the mean

scintillation counts obtained from the test groups relative to the corresponding mean

scintillation count obtained from controls.

The threshold level for the Stimulation Index to be considered a positive indicator of the

potential to cause skin sensitisation is 3.0.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The stimulation indices for the substance did not exceed the required value of 3 at any concentration tested.
Executive summary:

This study was conducted to assess the potential of the test article of the test article, to

cause skin sensitisation in the mouse.

Following a preliminary screening test using a 50% w/v formulation, the test article was

prepared for administration at 10, 25 and 50% w/v in dimethylformamide. Groups of five

female CBA I Ca mice were subjected to topical applications of vehicle or of one of the

test formulations to the outer aspect of the auditory pinnae once daily on Days I, 2 and 3.

On Day 6 a 20 muCi dose of tritiated 3H-methyl thymidine was injected intravenously into

each mouse. Five hours later the auricular lymph nodes were recovered from each

animal. The pairs of nodes from each animal were pooled and suspensions of the cellular

components of thelymph nodes were prepared in 5% w/v trichloroacetic acid and

processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices, the ratios of the mean

scintillation counts obtained from the test groups relative to the corresponding mean

scintillation count obtained from controls. The threshold level for the Stimulation Index

to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

The resulting Si were 0.73, 0.54 and 0.37 for 10%, 25% and 50% w/v respectively.

The Local Lymph Node Assay demonstrated that the test article does not have the

potential to cause skin sensitisation.

The test article did not meet the criteria for classification as a sensitiser according to EU

Commission Regulation (EC) 1272/2008.