Registration Dossier

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:
20 Dec 2019 - 12 Feb 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Palladium(II) acetate
EC Number:
222-164-4
EC Name:
Palladium(II) acetate
Cas Number:
3375-31-3
Molecular formula:
C2H4O2.1/2Pd
IUPAC Name:
palladium(2+) diacetate
Details on test material:
- Name of test material (as cited in study report): palladium(II)-acetate trimer
- Substance type: brown powder
- Physical state: solid
- Analytical purity: 47.15% palladium
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Purity test date: no data
- Lot/batch No.: 6597
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable throughout the test period “according to the sponsor”
- Storage condition of test material: no data
- Other:

Specific details on test material used for the study:
Palladium(II) acetate (>99.9% purity)
Pd content 48.22%

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
Nulliparous, non-pregnant female CBA/CaCrl strain mice 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.
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 8 to 15 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 16 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 10 weeks old on Day 1.

The animals were kept in the following conditions except for short periods of time where experimental procedures dictated otherwise:
-The animals were housed in groups of up to five during acclimatisation in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014). From Day-1, the preliminary study animal was individually housed and the main study animals were housed in groups of up to three.
-Bedding was provided on a weekly basis to each cage by use of clean European softwood 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 bedding at levels which might have interfered with achieving the objective of the study.
-Mains water was provided, ad libitum, via cage-mounted water bottles. The water had been periodically analysed for specific contaminants.
-No contaminants were present in water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.
-5LF2 EU Rodent Diet 14%, was freely available to the animals at all times. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer.
-No contaminants were present in diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.
-The animal rooms were designed to permit a minimum of 15 air changes per hour. The temperature and humidity ranges were 19 to 25C and 40 to 70% respectively. Daily recordings of maximum and minimum temperature and humidity were made.
-Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.
-In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and nesting materials. The nesting materials were removed from the cages the day before dosing.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
vehicle control, 10% w/v, 25% w/v, 50% w/v
No. of animals per dose:
5 female mice
Details on study design:

Preliminary screening:
- In the absence of toxicological information regarding irritation and/or systemic toxicity, a preliminary screening test was conducted with one animal.
The mouse was treated by daily application of 25 µL of the test article at the maximum suitable concentration (50% w/v in8 0% v/v acetone in
olive oill) to the dorsal surface of each ear for three consecutive days (Day 1, 2 and 3). The mouse was observed daily for any signs of toxicity or irritation at the application site. The body weight was recorded on Day-1 and prior to termination on Day 6. Both ears were observed for erythema and scored using a scale (0 - 4).
-Ear thickness measurements were taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6.
-Excessive local irritation is indicated by an erythema score ≥3 and/or an increase in ear thickness of ≥25% on any day of measurement.
-The animal was killed by an intraperitoneal injection of an overdose of sodium pentobarbitone at the end of the observation period. Death was confirmed by cervical dislocation.

Main study:
- Groups of five female mice were assigned to study according to the table given below. 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.
Group Number Group Description Concentration Number of Animals in Group
1 Vehicle control - 5
2 Test - low concentration 10% w/v 5
3 Test - intermediate conc 25% w/v 5
4 Test - high concentration 50% w/v 5
5 Positive control 25% v/v 5

 -The five groups of five female mice were subjected to application of the vehicle control, positive control 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 of3HTdR 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 the3HTdR, all mice were killed by exanguination under a deep plane of inhalation anasthesia. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.
- 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.
-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.
- Mice were weighed on Day-1 (the day before dosing) and on Day 6 prior to intravenous administration of3HTdR.
- Once death, the auricular lymph nodes were located and removed. The auricular lymph nodes of all mice from each dose group were placed into petri dishes containing 5 mL phosphate buffered saline.
- Incorporation of3HTdR 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The scintillation counter provided data including the DPM value (disintegrations per minute during a ten minute period) for each individual animal. The mean DPM value for each test group was divided by the mean DPM for the control group to provide the Stimulation Index (SI) value for each test group.

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).

Results and discussion

Positive control results:
The positive control article produced a Stimulation Index of 3.58, demonstrating adequate performance of the assay.

In vivo (LLNA)

Results
Key result
Parameter:
SI
Value:
> 3
Variability:
SI for 10%, 25% and 50% formulations were 15.74, 12.81 and 9.48, respectively
Test group / Remarks:
SI all well above the threshold value of 3.0, which is the threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation.
Remarks on result:
other: Positive indication of skin sensitisation

Any other information on results incl. tables

 group  DPM mean  DPM SD  P-value  SI
 control  12  5.5  /  /
 10%  195  80  0.0002  15.74
 25%  159  69.7  0.0017  12.81
 50% 118   51.3  0.016  9.48

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The Stimulation Indices with the test item were greater than the threshold of 3.0. Hence, the Local Lymph Node Assay demonstrated that Palladium(II) Acetate has the potential to cause skin sensitisation. The registrants consequently agree to classify the substance as Skin Sensit 1 (H317).
Executive summary:

This study was conducted to assess the potential of the test article, Palladium(II)

Acetate, to cause skin sensitisation in the mouse.

The in vitro methods described in OECD 442 C, D & E were found to be unsuitable

for use with Palladium(II) Acetate. The Local Lymph Node Assay was therefore

required to make a prediction of the skin sensitisation potential of the test article.

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 80% v/v acetone in olive

oil.

Groups of five female CBA/CaCrl mice were subjected to topical applications of

positive control, vehicle control or of one of the test formulations to the outer aspect

of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of

tritiated 3H-methyl thymidine was injected intravenously into each mouse.

Approximately 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 the lymph 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.

Observed Stimulation Indices of 15.74, 12.81 and 9.48 with concentration of Test Article in Applied Formulation (% w/v) of 10%, 25% and 50%, respectively, were observed. The positive control article produced a Stimulation Index of 3.58, demonstrating adequate performance of the assay.

Since the Stimulation Indices with the test item were greater than the threshold of 3.0, the Local Lymph Node Assay demonstrated that Palladium(II) Acetate has the

potential to cause skin sensitisation.

Palladium(II) acetate is consequently classified as Skin Sensitiser 1 (H317).