Mixed metal oxide complex

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 28 - June 16, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

impurity 1

Test material form:

solid: particulate/powder

Details on test material:

Batch No.: 2016JAN-15kg
Expiry Date: 01 January 2020
Appearance: Blue-white powder
Storage: Room temperature (15-25°C)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Specific details on test material used for the study:

Batch No.: 2016JAN-15kg
Expiry Date: 01 January 2020
Appearance: Blue-white powder
Storage: Room temperature (15-25°C)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Details on test system:

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item has an intrinsic colour (blue-white), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

Quality control:
EpiSkinTMSM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.
- Temperature of post-treatment incubation: After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, ≥95% humidified atmosphere.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

Following the formazan extraction, 2×200 µL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the Absorbance / Optical Density of the samples in a 96-well plate spectrophotometer at 570 nm using acidified isopropanol solution as the blank (6×200 µL).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
The test item was applied in its original form, no formulation was required. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface.

NEGATIVE AND POSITIVE CONTROL
A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface.

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.

Duration of post-treatment incubation (if applicable):

After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, ≥95% humidified atmosphere.

Number of replicates:

Three replicates were used for the test item and positive and negative controls respectively. Furthermore, two additional test item treated replicates were used for colour controls.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

> 99 - < 100

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Pre incubation:

The “maintenance medium” was pre-warmed to. The appropriate number of an assay plate wells were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight atin an incubator with 5 % CO_2,≥95% humidified atmosphere.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Neodymium fluoride oxide, magnesium doped is considered to be non-irritant to skin and these results should not drive any GHS classification.

Executive summary:

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO₂. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution atin 5% CO₂ protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.

The test item has an intrinsic colour (blue-white), therefore two additional test item treated tissues were used for the non-specific OD evaluation.

SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

 

In this in vitro skin irritation test using the EPISKIN model, the test item Neodymium fluoride oxide, magnesium doped did not show significantly reduced cell viability in comparison to the negative control(mean value: 99 %). Allobtained test item viability results were above or equal to 50 % when compared to the viability values obtained from the negative control. The test item was considered to be non-irritant to skin.