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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2011-12-08 to 2011-10-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recent GLP compliant study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
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:
N-butyl-2,2,6,6-tetramethylpiperidin-4-amine, oligomeric reaction products with N,N'-bis(3-aminopropyl)ethylenediamine and 2,4,6-trichloro-1,3,5-triazine
EC Number:
500-311-6
EC Name:
N-butyl-2,2,6,6-tetramethylpiperidin-4-amine, oligomeric reaction products with N,N'-bis(3-aminopropyl)ethylenediamine and 2,4,6-trichloro-1,3,5-triazine
Cas Number:
120498-03-5
IUPAC Name:
1,3-Propanediamine,N-N’’-1,2-ethanediylbis- reaction product with 1,3,5-triazine-2,4-diamine,N,N’-dibutyl-6-chloro-N,N’-bis(2,2,6,6-tetramethyl-4-piperidinyl), polymer with N-butyl-4,6-dichloro-N-(2,2,6,6-tetramethyl-4-piperidinyl)-1,3,5-triazin-2-amine
Details on test material:
The test article, a white powder, was identified as Uvasorb HA 88 and was received at Covance as follows:
Test article CAS number Batch number Quantity supplied (g) Purity (%) Expiry Date Date of receipt at Covance

Uvasorb HA 88 136504-96-6 SD1004-225A1 580.3 Not stated Not stated 13 October 2011


The purity and an expiry date for the test article were not stated by the Sponsor.
When not in use the test article was stored in a sealed container, at room temperature (15 to 25°C), in the dark.
The vehicle for the test article was 80% v/v acetone in olive oil, prepared by Covance from acetone supplied by VWR International Ltd, and olive oil supplied by Sigma-Aldrich Co. Ltd. The vehicle was chosen because it was the first of the listed vehicles that produced an overtly stable solution.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Nulliparous, non-pregnant female CBA/CaOlaHsd strain mice were obtained from Harlan UK Limited, Bicester. All animals were given a clinical inspection for ill health on arrival and a sample was weighed.
Justification for selection of the test system: The sex and strain of mouse has been selected for this study in conformance with the design of the validated test method (OECD Guidelines for Testing of Chemicals Method 429, adopted 24 April 2002).

Acclimatisation period and allocation to study: Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. The condition of the animals was assessed daily throughout the acclimatisation period of 7 to 14 days. A second inspection was performed prior to study commencement to ensure the animals were suitable for the study. Overtly healthy animals were arbitrarily allocated to the study groups on the day prior to commencement of treatment.
Animals in the main study were in a body weight range of 18 to 22 g on the day before dosing commenced. Individual body weights were within ± 20% of the mean body weight for mice on the study. Based on information from the supplier the mice were approximately 9 to 11 weeks old on Day 1.

Caging: The animals were housed in groups of up to five animals during acclimatisation and individually housed from Day –1 in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Used in Scientific Procedures' (Home Office, London, 1989).

Diet, water and bedding: SQC(E) Rat and Mouse Maintenance Diet No 1, from Special Diets Services Ltd, Witham, UK was freely available to the animals at 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 water bottles. The water had been periodically analysed for specific contaminants.
Certificates of analysis for contaminant levels in each 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 of clean European soft wood bedding (Datesand Ltd, Manchester, UK). The bedding had been analysed for specific contaminants and the results retained on file at Covance.
No contaminants were present in diet, water or bedding at levels which might interfere with achieving the objective of the study.

Environment: The animal rooms were designed to permit 15 to 20 air changes per hour. The temperature and humidity ranges were 20 to 24C and 45 to 65% respectively. Daily recordings of maximum and minimum temperature and humidity were made. The relative humidity in the animal room was recorded below the protocol range on one occasion, at 40%. This deviation does not affect the integrity of the study. The rooms were illuminated by fluorescent strip-lights for twelve hours daily.

Identification of the test system: A unique number inscribed onto the tail with indelible ink on the day prior to dosing individually identified the mice. A colour coded card on each cage gave information including study number, animal number and sex.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 10, 25 and 50% w/v
No. of animals per dose:
Four females per dose group and control
Details on study design:
Preliminary screening test:
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test article, 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 80% acetone in olive oil) 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 of treatment. Any signs of toxicity or irritation during this period were recorded. The body weight was recorded on Day –1. The animal was killed by exposure to a rising concentration of carbon dioxide at the end of the observation period.
Constitution and function of groups:
Groups of four female mice were assigned to study according to the table given below. Doses were selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1.0%, 0.5% etc. Three consecutive concentrations were selected on the basis of the preliminary screening test so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irritation.
Main study 1
Group number Group description Concentration Number of animals
in group
1 Vehicle control - 4
2 Test – low concentration 10% w/v 4
3 Test – intermediate concentration 25% w/v 4
4 Test – high concentration 50% w/v 4

The main study was repeated at the same concentrations stated above due to the control values being low and possibly skewing the results of the test article treated groups.
Main study 2
Group number Group description Concentration Number of animals
in group
5 Vehicle control - 4
6 Test – low concentration 10% w/v 4
7 Test – intermediate concentration 25% w/v 4
8 Test – high concentration 50% w/v 4

Formulation of test articles and tritiated thymidine:
Formulations were freshly prepared as required using 80% v/v acetone in olive oil on Days 1, 2 and 3 of both main study phases. The formulations were stored at room temperature, in sealed, air-tight containers prior 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 of test article were expressed volumetrically and in terms of test article received (without regard to purity or active content).
Tritiated 3H-methyl thymidine (3HTdR), batch 201106, was obtained as a 1000 μCi/mL preparation from PerkinElmer Life and Analytical Sciences, Boston, USA. An aliquot of 5.75 mL phosphate buffered saline was mixed with 0.5 mL 3HTdR shortly before intravenous dosing commenced on Day 6. The final product contained 3HTdR 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 was treated by direct application of the appropriate test or control formulation (0.025 mL/pinna) dispensed from an automatic micro pipette.
Treatment regimen
The eight groups of four female mice were subjected to application of the vehicle or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3.
On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR into a tail vein of 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 rising concentration 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.

EXPERIMENTAL OBSERVATIONS
Clinical signs
Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article.
Routine health checks
All animals were examined at the beginning and end of the working day throughout the acclimatisation and study periods to ensure they were in good health.
Body weights
Mice were weighed on Day 1 (the day before dosing) and on Day 6 prior to intravenous administration of 3HTdR.

TERMINAL PROCEDURES - RECOVERY OF LYMPH NODES
Once death had been confirmed each mouse was placed on a bench lined with paper with its ventral aspect uppermost. A mid 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 from the capsule of the nodes. The auricular lymph nodes of all mice from each dose group were placed into petri dishes containing 5 mL phosphate buffered saline.
Preparation for scintillation count
The lymph nodes collected into each petri dish were cut open and disaggregated by squashing the fragments with a sharp blade. The resultant liquor was transferred into code identified conical tubes. The petri dishes were rinsed with an additional 5 mL phosphate buffered saline and the second liquor was added to the first liquor. At each transfer debris, such as fragments of capsule, were retained in the petri dish wherever possible.
After 5 minutes the pooled liquor was filtered into a second conical tube by transferring the liquor into a 10 mL syringe and passing it through a 200 µm mesh stainless steel gauze containing a fabric filter, cut to size (Clarcor UK, Lockertex Filtration Products, Warrington, UK). Any visible sediment remaining 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 was centrifuged at 190 G for 10 minutes. The supernatant was discarded and the pellet resuspended in 3 mL of 5% w/v aqueous trichloroacetic acid. The suspension was stored for 18 hours at 2 to 8C (nominal 4°C).
On the following day the suspension was re-centrifuged at 190 G for 10 minutes and the supernatant was drawn off and discarded. The pellet was resuspended in 1 mL 5% w/v aqueous trichloroacetic acid, then subjected to ultrasonic dispersion for 25 minutes to ensure an homogenous suspension. The suspension (1 mL) was transferred to a scintillation vial and scintillation fluid (ca 10 mL) was added.
Scintillation counting
Once prepared the scintillation vials were placed in the appropriate carrier racks. Two background vials were prepared, one containing ca 10 mL of scintillation fluid and the other containing 1 mL of 5% w/v aqueous trichloroacetic acid and ca 10 mL scintillation fluid. The carrier rack was passed into the scintillation counter. All vials, including the background samples, were submitted for liquid scintillation counting for 10 minutes, using a 3H quench curve.
Incorporation of 3HTdR is measured by ß-scintillation counting as disintegrations per minute (DPM) over a ten-minute period. This value was corrected to account for the background containing 5% w/v aqueous trichloroacetic acid and scintillation fluid.

DATA EVALUATION
The scintillation counter printed data including the DPM value (disintegrations per minute during a ten minute period). The DPM value was transformed into a DLM value (disintegrations per minute per lymph node) by dividing by the number of sites yielding lymph nodes. The DLM value for each test group was divided by the DLM for the control group to provide the Stimulation Index (SI) value for each test group.
Criteria for assessment of test results
The test result is not valid for those groups producing an SI value of 3.0 or more when the sites of application have shown excessive irritation and for those groups that have shown indications of systemic toxicosis.
The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above.
The test article is classified as a non sensitiser when the maximum value of the SI is less than 3.0. (This result is unchanged by observations of irritation at sites of application of the test formulation).

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Stimulation Index calculation

Results and discussion

Positive control results:
Demonstrate sensitive test system

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation count per group obtained from the test groups relative to the corresponding mean scintillation count 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.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Table 2 Scintillation counts and stimulation indices Main Study 1 Sample identity Number of sites yielding lymph nodes Disintegrations per minute* (DPM) Disintegrations per minute per node (DLM) Stimulation Index (SI) Scintillation fluid with 5% w/v trichloroacetic acid -- 62 -- -- Vehicle control 8 35 4 -- Test article, 10% w/v 8 171 21 5.3 Test article, 25% w/v 8 172 22 5.5 Test article, 50% w/v 8 272 34 8.5 Main Study 2 Sample identity Number of sites yielding lymph nodes Disintegrations per minute* (DPM) Disintegrations per minute per node (DLM) Stimulation Index (SI) Scintillation fluid with 5% w/v trichloroacetic acid -- 62 -- -- Vehicle control 8 179 22 -- Test article, 10% w/v 8 547 68 3.1 Test article, 25% w/v 8 950 119 5.4 Test article, 50% w/v 8 563 70 3.2 * All scintillation counts corrected for the blank SI = Test group DLM value Control group DLM value

Any other information on results incl. tables

Death or signs of systemic toxicity/excessive irritation were not noted.

From Day 2 onwards, hard clumps of fur on the back of the neck were noted.

Based on this information the dose levels selected for the main test were 50%, 25% and 10% w/v in 80% acetone in olive oil.

                

All animals survived treatment.

Clinical signs:

The vehicle and test formulation application sites remained free of irritation.

On Day 1, greasy wet fur to the back of the neck was noted in all animals. Greasy fur to the top of the head was also noted in all animals in main study 2.

On Day 2 onwards, greasy wet fur to the back of the neck was noted in all animals in main study 1, together with hairloss to the back of the neck in animals in Groups 3 and 4 in main study 1.

On Day 2, crusty fur to the neck and ears was noted in Groups 7 and 8 of main study 2. Also noted from Day 2 onwards was greasy fur to the neck and top of the head in all animals in main study 2.

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


Stimulation Index :

Main Study 1

 

Concentration of test article in applied formulation (% w/v)

10%

25%

50%

Stimulation Index

5.3

5.5

8.5

 

Main study 2

 

Concentration of test article in applied formulation (% w/v)

10%

25%

50%

Stimulation Index

3.1

5.4

3.2


Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The Local Lymph Node Assay demonstrated that Uvasorb HA 88 has the potential to cause skin sensitisation.
The test article was classified as a Category 1 sensitiser according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).