Disodium tetrachloropalladate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 May 2018 - 11 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

adopted 25 June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No. 640/2012 of 6 July 2012 amending Regulation (EC) No. 440/2008; EC method B.51: Skin Sensitisation, Local Lymph Node Assay: BrdU-ELISA.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 002

Cas Number:

13820-53-6

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): sodium chloropalladite
- Substance type: brown powder
- Physical state: solid
- Analytical purity: no data
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Purity test date: no data
- Lot/batch No.: 041118/A
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: supplied in closed glass jar; no further details

Specific details on test material used for the study:

99.9% Na2PdCl4 (based on Pd content of 36.14%)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:JN

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier Labs, CS 4105 Le Genest, Saint Isle, Saint Berthevin, Cedex 53941, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at main study initiation: approx 8 weeks
- Weight at study initiation: 19-23 g
- Housing: Before application the animals were housed in groups of 5 animals in MAKROLON cages (type III) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 15 cm. After application the animals were housed singly in order to prevent them licking off the test item from the ears of the other animals. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material. The cages were changed and cleaned once a week.
- Diet: Commercial diet ssniff® R/M-H V1534 (sourced from ssniff Spezialdiäten GmbH, 59494 Soest, Germany) was offered ad libitum. Food residue was removed.
- Water: Tap water was offered ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3 (maximum range)
- Humidity (%): 55 ± 15 (maximum range)
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12 (about 150 lux at approx 1.5m room height)

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Remarks:

Acetone/olive oil (4:1, v/v) and N,N-dimethylformamide, other recommended vehicles, did not provide higher concentrated suitable suspensions. Dimethyl sulfoxide was not considered as vehicle at the request of the Sponsor.

Concentration:

Pre-screening test: 10, 25, 50 and 75% (w/w)
In the preliminary test employing two animals per dose, concentrations of 10% or 25% (w/w) showed irritating properties in form of an erythema score ≥ 3 and/or an ear thickness increase of more than 25% which is evidence of irritating properties.
Main study: 0, 1, 2.5 and 5% (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN (RANGE-FINDING) TESTS:
- Compound solubility: In a preliminary solubility assessment, propylene glycol was selected as it is recommended by the OECD guideline and provided suitable solutions of the test item both for administration and adherence to the mouse ear of such high concentrations. Doses were selected based on the recommendations in the OECD guideline, adjusted to the physical appearance of the test item as solid, with 75, 50, 25 and 10% (w/w). A 25% concentration was the highest concentration of the test item in propylene glycol to obtain an applicable solution.
- Test item adminstration: The test item formulations were administered to the dorsum of both ears of each animal at an application volume of 25 µL/ear.
- Systemic toxicity and Irritation: All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination (Day 6). Both ears of each mouse were observed for erythema and scored on a 0-4 scale (0: no erythema; 1: very slight erythema (barely perceptible); 2: well-defined erythema; 3: moderate to severe erythema; 4: severe erythema (beet redness) to eschar formation preventing grading of erythema).
The following clinical observation would indicate systemic toxicity: changes in nervous system function (e.g. pilo-erection, ataxia, tremors, and convulsions); changes in behaviour (e.g. aggressiveness, change in grooming activity, marked change in activity level); changes in respiratory patterns (i.e. changes in frequency and intensity of breathing such as dyspnoea, gasping, and rales), and changes in food and water consumption. In addition, signs of lethargy and/or unresponsiveness and any clinical signs of more than slight or momentary pain and distress, or a >5% reduction in body weight from Day 1 to Day 6 and mortality would be considered in the evaluation. Moribund animals or animals showing signs of severe pain and distress would have been humanely killed.
- Ear thickness measurements: 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. Additionally, on Day 6, ear thickness was determined by ear punch weight determinations, which was performed after the animals were humanely killed. Excessive local irritation is indicated by an erythema score ≥3 and/or ear thickness increase of ≥25% on any day of measurement. The highest dose selected for the main LLNA: BrdU-ELISA study was the next lower dose in the pre-screen concentration series that doses not induce systemic toxicity and/or excessive local skin irritation.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay BrdU-ELISA
- Criteria used to consider a positive response: Results of each treatment group are expressed as the mean SI. The SI is derived by dividing the mean BrdU luminescence/mouse within each test item group and the positive control group by the mean BrdU luminescence labelling index for the solvent/vehicle control group. The average SI for the vehicle control is then one.
SI = (Mean test item or positive control group)/(Mean negative control group)
The decision process regards a result as positive when SI ≥ 1.6. However, the strength of the dose-response relationship, the statistical significance and the consistency of the vehicle and positive control responses would also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) is declared positive.
For a borderline positive response between an SI of 1.6 and 1.9, it may be necessary to consider additional information such as dose-response relationship, evidence of systemic toxicity or excessive irritation, and where appropriate, statistical significance together with SI values to confirm that such results are positives. Consideration would be also given to various properties of the test item, including whether it has a structural relationship to known skin sensitizers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response observation.
In addition, the average ear weights per group and the average ear thickness per group were compared to the vehicle control group as an indication for possible irritating properties.

TREATMENT PREPARATION AND ADMINISTRATION: g
•Day 1: The weight of each animal was identified and recorded. Ear thickness of left and right ear of each animal was recorded. Any clinical observation was recorded. 25 µL of the appropriate dilution of the test item, the negative control, the positive control or the vehicle of the positive control were applied to the dorsum of each ear.
•Days 2 and 3: The application procedure carried out on day 1 was repeated.
•Day 4: No treatment.
•Day 5: 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected inter-peritoneally.
•Day 6: The weight of each animal and any clinical observation were recorded. Approximately 24 hours after BrdU injection the animals were humanely killed. The draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline (PBS) for each animal. To further monitor the local skin response in the study, additional parameters such as scoring of ear erythema and ear thickness measurements (obtained by using a thickness gauge and ear punch weight determinations at necropsy) were carried out.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Since a concentration-related increase in values was observed, a comparison to negative control was carried out using Dunnet's test.
In case of heterogeneity of variances, the Student's t-test is carried out, limit of significance is p ≤ 0.01.
Dose-response were analyzed according to Pearson's correlation test.

Results and discussion

Positive control results:

Treatment with the positive control item caused a statistically significant increase in the BrdU labelling index, compared to the negative control (propylene glycol), which reflects a positive sensitisation response. The Stimulation Index of 1.665 exceeded the threshold value of 1.6 over the positive control vehicle group, and therefore, the study can be regarded as valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.111

Test group / Remarks:

1%

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Remarks:

Treatment with the test item at concentrations of 1, 2.5 and 5% (w/w) revealed a dose-related increase in the BrdU labelling index (SI values of 1.111, 1.310 and 1.881, respecitvely). The SI value of the highest dose thus exceeded the threshold of 1.6.

Results 2

Parameter:

SI

Value:

1.31

Test group / Remarks:

2.5%

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Remarks:

Treatment with the test item at concentrations of 1, 2.5 and 5% (w/w) revealed a dose-related increase in the BrdU labelling index (SI values of 1.111, 1.310 and 1.881, respecitvely). The SI value of the highest dose thus exceeded the threshold of 1.6.

Results 3

Key result

Parameter:

SI

Value:

1.881

Test group / Remarks:

5%

Remarks on result:

positive indication of skin sensitisation based on QSAR/QSPR prediction

Any other information on results incl. tables

In a preliminary test employing two animals per dose, concentrations of 10% or 25% (w/w) showed irritating properties in form of an erythema score ≥ 3 and/or an ear thickness increase of more than 25% which is evidence of irritating properties.

Parameter	Negative control	1% test item	2.5% test item	5% test tem	Positive control	Vehicle of positive control
BrdU labelling index	3.009	3.343	3.943*	5.660*	5.377*	2.99
Stimulation index (SI)	1.000	1.111	1.310	1.881	1.793	1.000
Ear weight (mg)	14.4	14.2	14.9	17.4*	16.7	14.8
Difference of ear thickness (mm, TD6)	220.0	222.0	227.0	243.0	240.0	224.0

  * statistically significant increase compared to negative control at p ≤ 0.01

The stimulation indices calculated for the BrdU labelling index of the animals treated with the 1 or 2.5% concentrations did not exceed the threshold value of 1.6. At the highest concentration (5% w/w) there was a dose related increase in the BrdU Labelling index and the stimulation index was 1.881, which exceeded the threshold value of 1.6.

There were no deaths and no systemic clinical signs or effects on body weights were observed during the study. At the highest concentration (5% w/w) there was a dose related increase in ear weight (punch biopsies) comparable to that seen in the positive control group.The ear thickness on test day 6 compared to the start values was not or only slightly increased.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Under the present test conditions, the Stimulation Index (SI) of the BrdU labelling index exceeded the threshold-value of 1.6 at the highest concentration of 5%(w/w) (in this case SI = 1.881) and no obvious signs of irritation were observed. This borderline response may indicate skin sensitizing properties of the test item Disodium Tetrachloropalladate.

Executive summary:

The skin sensitising potential of disodium tetrachloropalladate has been assessed in a GLP mouse local lymph node assay (LLNA): BrdU-ELISA, conducted according to OECD Test Guideline 442B. Following a preliminary range-finding study to assess irritancy, female mice CBA/JN mice (5/group) were treated topically with 0, 1, 2.5 and 5% test item (in propylene glycol) on three consecutive days. There was no treatment on test days 4, 5 and 6. On day 6, cell proliferation in the local lymph nodes was measured by incorporation of injected 5-bromo-2-deoxyuridine (BrdU) using ELISA. The cell proliferation in the local lymph nodes was determined by measuring the BrdU content with BrdU-ELISA and the values obtained were used to calculate Stimulation Indices (SI).

The stimulation indices calculated for the BrdU labelling index of the animals treated with the 1 or 2.5% concentrations did not exceed the threshold value of 1.6. At the highest concentration (5% w/w), there was a statistically significant increase in the BrdU labelling index and the stimulation index was 1.881. This value exceeds the threshold value of 1.6 and is indicative of a borderline positive response.

There were no deaths and no systemic clinical signs or effects on body weights were observed during the study. At the highest concentration (5% w/w) there was a dose related increase in ear weight (punch biopsies), which was comparable to that seen in the positive control group.The ear thickness on test day 6 compared to the start values was not or only slightly increased, i.e. no skin irritation properties were noted.

Treatment with the positive control item caused the expected increases in the BrdU labelling index compared to the negative control, and a Stimulation Index of 1.793, which was in excess of the threshold value of 1.6. Therefore, the study can be regarded as valid.

In conclusion, under the present test conditions, the test item Disodium Tetrachloropalladate, showed a positive response. The Stimulation Index (SI) of the BrdU labelling index exceeded the threshold-value of 1.6 at the highest concentration of 5%(w/w) (in this case SI = 1.881) and no obvious signs of irritation were observed. This may indicate to skin sensitizing properties of the test itemDisodium Tetrachloropalladate.