Dichromium nitride

Endpoint summary

Description of key information

Skin irritation: not irritating (OECD 439; GLP compliant)

Eye irritation: not irritating (OECD 405; GLP compliant)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-20 to 2017-12-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2012-07-06

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2014-11-07

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, keep dry in closed container

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

other: Dulbecco's phosphate buffered saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 25867

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- if the mixture turns blue/purple, the test item is presumed to have reduced MTT and the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using freeze-killed tissues.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item was mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- if the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential using additional living tissues treated with the test item.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 ± 2 h.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate was shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. Optical density (OD) was measured with a filter band without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponds to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [mean ODtest item / positive control / ODmean of negative control] * 100
Irritant potential of the test item will be predicted from the relative mean tissue viabilities compared to the negative control tissues. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (39 mg/cm²) of the test item

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL of the vehicle

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % sodium dodecyl sulfate solution (TC-SDS-5 %)

Duration of treatment / exposure:

60 ± 1 minute

Duration of post-treatment incubation (if applicable):

approx. 42 hours

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Remarks:

(mean)

Value:

97

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.
- Colour interference with MTT: mixture of 25 mg of the test item per 300 µl aqua dest. and/or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.865).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.1%)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2 % - 5.2 %).

Please also refer to the field "An other information on results incl. tables" below.

Table 1: Result of the test Item dichromium nitride

Name	Negative Control (NK)			Positive Control (PC)			Test Material (TM)
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.956	1.874	1.856	0.096	0.102	0.099	1.780	1.748	1.942
	1.969	1.899	1.889	0.097	0.103	0.102	1.815	1.837	1.987
OD570(Blank Corrected)	1.913	1.832	1.814	0.053	0.059	0.057	1.737	1.706	1.899
	1.926	1.856	1.846	0.055	0.061	0.059	1.773	1.795	1.945
Mean OD570of the Duplicates (Blank Corrected)	1.920	1.844	1.830	0.054	0.060	0.058	1.755	1.750	1.922
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.865*			0.057			1.809
SD OD570	0.048			0.003			0.098
Relative Tissue Viability [%]	103.0	98.9	98.1	2.9	3.2	3.1	94.1	93.9	103.1
Mean Relative Tissue Viability [%]	100.0			3.1**			97.0
SD Tissue Viability [%]***	2.6			0.2			5.2
CV [% Viabilities]	2.6			5.4			5.4

^*               Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^**         Mean relative tissue viability of the three positive control tissues is£ 20%.

^***        Standard deviation (SD) obtained from the three concurrently tested tissues is≤ 18%

Table 2: Historical data

	Mean OD570±30nm Negative control (NK)	Mean Relative Viability [%] Positive control (PC)	SD Viability [%]
Mean	1.843	4.3	4.2
SD	0.286	2.2	4.7
n	22	22	84

Historical data were generated from 2015 to 2017.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not irritating to the skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-12 to 2018-02-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2017-05-08

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10 °C to +25 °C, in the closed original container in a dry, well-ventilated place.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
- Age at study initiation: approx. 7 - 8 months
- Weight at study initiation: 3.7 to 4.7 kg
- Housing: kept singly in cages with dimensions of 823 mm x 660 mm x 500 mm (Scanbur, Denmark)
- Diet (ad libitum): commercial diet, ssniff® K-H V2333 (ssniff Spezialdiäten GmbH)
- Water (ad libitum): tap water
- Acclimation period: at least 20 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 °C ± 3 °C (maximum range)
- Relative humidity: 30 % - 70 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg of the test item

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

1, 24, 48 and 72 hours after the administration

Number of animals or in vitro replicates:

3 male rabbits

Details on study design:

INITIAL AND CONFIRMATORY TEST
The test was performed initially using one animal. As no corrosive or severe irritant effects were observed in the animal treated in the preliminary test, 2 further animals were employed 24 hours after start of the initial test.

USAGE OF ANAESTHETICS AND SYSTEMIC ANALGESICS
Sixty minutes prior to test item administration, 0.01 mg Buprenovet®/kg bw were administered by subcutaneous (s.c.) injection to all animals to provide a therapeutic level of systemic analgesia to avoid or minimize pain and distress.
Five minutes prior to the test item administration, two drops of Conjuncain®EDO, a topical anaesthetic, were applied to each eye of all animals, to the right eye, in which the test item was to be applied, and to the left eye, which served as control.
Eight to 9 hours after administration, Acticam® 0.5 mg/kg, s.c. was administered to provide a continued therapeutic level of systemic analgesia.

REMOVAL OF TEST SUBSTANCE
- Washing: eyes were rinsed with 20 mL 0.9 % aqueous NaCl solution
- Time after start of exposure: 1 hour

SCORING SYSTEM: according to the Draize scale and a scale scoring the degree of staining (Fluorescein-test)

TOOL USED TO ASSESS SCORE: slit lamp and fluorescein
The eyes were examined ophthalmoscopically with a slit lamp prior to the administration and 1, 24, 48 and 72 hours after the administration. The eye reactions were observed and registered.
24, 48 and 72 hours after administration, fluorescein was applied to the eyes before being examined to aid evaluation of the cornea for possible lesions.

OBSERVATIONS
- body weight: at the beginning and at the end of the study.
- behaviour and food consumption were monitored
- any adverse systemic effects were recorded

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other: slight corneal opacity was observed at the 1 hour observation.

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

- slight corneal opacity (grade 1) was observed in one animal 1 hour after administration. The effect was fully reversible within 24 hours after application of the test item.
- fluorescein test performed 24, 48 and 72 hours after administration did not reveal any changes.
- irises and the conjunctivae were not affected by administration of the test item.

Other effects:

- Lesions and clinical observations: no systemic intolerance reactions were observed.

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating to the eyes.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation

The substance was not observed to be irritating to the skin in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation

The substance was tested for its eye irritation properties according to OECD 437, but the result cannot be used for classification since the result was equivocal. Furthermore, an in vivo eye irritation test according to OECD 405 (2017) was conducted. The results showed that the substance was not irritating to the eyes.

Justification for classification or non-classification

Skin irritation

The substance does not possess an skin irritating potential based on an in vitro OECD 439 (2015) test and does not require classification as skin irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation

The substance does not possess an eye irritating potential based on an in vivo OECD 405 (2017) test and does not require classification as eye irritating according to Regulation (EC) No 1272/2008 and its subsequent adaptations.