Potassium niobate

Endpoint summary

Description of key information

Two in vitro studies were conducted according to OECD 442E and 442D respectively. No solvent compatible with the KeratinoSens was found to dissolve the test item in the required concentration (200 mM in dimethyl sulfoxide (DMSO), water or tetrahydrofuran (THF)). Therefore, the study could not be performed.

In a dermal sensitization study conducted according to OECD 429 with Potassium niobate ( ≥ 99.8 % purity) dissolved in DMSO, young adult female CBA/CaOlaHsd mice (5 per dose group) were tested at concentrations of 12.5% (v/v), 25 % (v/v) and 50 % (v/v) in a local lymph node assay (LLNA). Due to animal welfare reasons the negative control was shared and a periodically performed positive control (1% Phenlyenediamine) was used. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 39.23%. In this study, the test item is a dermal sensitizer under "Category 1B" according to UN GHS.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-08 to 2018-04-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

adopted 22nd July, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 10–11 weeks
- Housing: The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Yes, Altromin 1324 maintenance diet
- Water (e.g. ad libitum): Yes, tap water
- Acclimation period: at least 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

dimethyl sulphoxide

Concentration:

12.5%, 25% and 50% (w/v), suspended in DMSO

No. of animals per dose:

5 females per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals. The maximum technically applicable concentration of the test item in the vehicle DMSO was found to be 50%.

- Irritation: In order to determine the highest tolerated and not excessively irritant test concentration a pre-screen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The 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. Both ears were observed for erythema and scored. Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%. Four animals were treated by topical application with the test item on three consecutive days at the following concentrations to the entire dorsal surface of each ear:
Animals no. 1 and no. 2 were treated with a test item concentration of 50% (suspended with DMSO, Sigma-Aldrich, lot no. SHBF2742V, expiry date:17/03/2018) Animals no. 3 and no. 4 were treated with a test item concentration of 25% (suspended with DMSO) One further animal was treated with 100% DMSO and served as negative control (animal no. 5).

- Systemic toxicity: During this period also all clinical signs were recorded. Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).

- Ear thickness measurements: Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured

- Erythema scores: See Table 1 in "Any other information on results". For the results of the pre-screen tests please refer to the box "Any other information on results".

MAIN STUDY

Control
DMSO was used as vehicle and served as negative control. For animal welfare reasons the negative control was shared.

Other Materials
3H-methyl thymidine (TRK 300, 20 Ci/mmol; PerkinElmer, lot no. 201710E), diluted to a working concentration of 80 µCi/mL.
Phosphate buffered saline (PBS), BSL Munich, lot no. 240118, expiry date: 09/2018.
Trichloroacetic acid (TCA), Sigma-Aldrich, lot no. BCBT4970V, expiry date: 19/05/2018.

Preparation of the Test Item
Based on the results observed in the pre-screen test the following test item concentrations were selected for the main study:
12.5%, 25% and 50% (w/v), suspended in DMSO. The preparations were made immediately prior to each dosing.

Preparation of the Animals
The animals were randomly selected using the validated departmental computerised system E WorkBook (latest version, ID Business Solutions Ltd.).
Identification was ensured by cage number and individual marking (tail).

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Test Regime
Topical Application: Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine: Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension: Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine: The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3= c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA, if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/201 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, seventh revised edition, 2017:
Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%
WARNING, exclamation mark. May cause an allergic skin reaction.

Positive control substance(s):

other: 1 % Phenylenediamine

Positive control results:

The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system (see Table 2 in box "Any other information on results").

Key result

Parameter:

SI

Remarks:

Mean of five animals

Value:

4.7

Variability:

S.D = 1.4

Test group / Remarks:

50 % (v / v)

Key result

Parameter:

SI

Remarks:

Mean of five animals

Value:

0.8

Variability:

S.D = 0.3

Test group / Remarks:

25 % (v / v)

Key result

Parameter:

SI

Remarks:

Mean of five animals

Value:

0.7

Variability:

S.D = 0.1

Test group / Remarks:

12.5 % (v / v)

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Please see Table 3 in box "Any other information on results"

EC3 CALCULATION
The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 39.23%.

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any clinical signs

BODY WEIGHTS
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

WEIGHT OF LYMPH NODES
The means of the lymph node weights per group showed relevant difference only for the 50% test group compared to the negative control.
The mean weight of the lymph nodes
for the 50% test group was 5.6 mg
for the 25% test group was 4.0 mg
for the 12.5% test group was 4.0 mg
for the negative control group was 4.1 mg

Results of the pre-screen tests

Compound Solubility: The maximum technically applicable concentration of the test item in the vehicle DMSO was found to be 50%.

Irritation: Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal. All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the prescreen test.

Results of the main study

Table 2: Stimulation Indices obtained for the Positive-Control Group

Test item	Conc. (%)		Stimulation index
Negative control DMSO	100	Mean value of five animals	1.0
		S.D
Positive control Phenylenediamine in DMSO	1	Mean value of five animals	7.8
		S.D	2.7

Table 3: Stimulation Indices obtained in the main experiment

Test item	Conc. (%)		Stimulation index
Negative control DMSO	100	Mean value of five animals	1.0
		S.D
Potassium niobate in DMSO	50	Mean value of five animals	4.7
		S.D	1.4
Potassium niobate in DMSO	25	Mean value of five animals	0.8
		S.D	0.3
Potassium niobite in DMSO	12.5	Mean value of five animals	0.7
		S.D	0.1

SD = standard deviation; MV = mean value

If not noted individually, the results include both lymph nodes of an animal.

Interpretation of results:

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

Conclusions:

In conclusion, in a mouse local lymph node assay, the test item is considered to be a skin sensitizer (Skin Sens. 1B) with an EC3 value of 39.23%.

Executive summary:

In a dermal sensitization study conducted according to OECD 429 with Potassium niobate ( ≥ 99.8 % purity) dissolved in DMSO, young adult female CBA/CaOlaHsd mice (5 per dose group) were tested at concentrations of 12.5% (v/v), 25 % (v/v) and 50 % (v/v) in a local lymph node assay (LLNA). Due to animal welfare reasons the negative control was shared and a periodically performed positive control (1% Phenlyenediamine) was used. Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal. No mortality was observed in any of the animals. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 39.23%. The results of the radioactivity determination are supported by the second endpoint, the means of the lymph node weights per group, which showed increased values for the test group exceeding the stimulation index of 3 compared to the negative control values and similar values for both other tested concentrations that did not exceed the stimulation index of 3 compared to the negative control values. In this study, the test item is a dermal sensitizer under "Category 1B" according to UN GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the result from a dermal sensitization study conducted according to OECD 429, Potassium niobate needs to be classified as Skin Sens 1B, H317 according to CLP regulation 1272 / 2008.