Chromium diboride

Endpoint summary

Description of key information

In an in vitro skin irritation assay conducted in accordance with OECD test guideline 439, Chromium diboride (> 99.4% purity) was applied topically to a reconstituted three-dimensional human epidermis. Based on the results, the target substance was considered to be non-irritating to the skin.

The eye irritation potential of Chromium diboride (>99.4% purity) was examined in an in vivo study according to OECD 405. Only a slight irritant effect (grade 1) which was fully reversible within 48 hours could be observed. Based on the results the target substance can be considered as non-irritant to the eye.

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:

2016-08-09 to 2016-09-29

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

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

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM™ (SkinEthic)
- Tissue batch number(s): 16-EKIN-035

SkinEthic Kit:
- EPISKIN-SM™ plate containing 12 reconstructed epidermis units (area: 0.38 cm²); each reconstructed epidermis is attached to the base of a tissue culture insert with an O-ring set and maintained on nutritive agar for transport (Lot No.: 16-EKIN-035):
1x 12-well assay plate
1x flask of sterile maintenance medium (basic medium for incubations, Lot No.: 16-MAIN3-056 / 16-MAIN3-060)
1x flask of sterile assay medium (basic medium for use in MTT assays, Lot No.: 16-ESSC-038)

- Validity controls as provided by the supplier (SkinEthic):
Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
Specification ≥ 19.5
Result: 22.5 ± 0.4, CV = 2.0%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
Barrier function:
IC50 determination (SDS concentration, MTT test, n= 14):
Specification ≥ 1.5 mg/mL
Result: 1.8 mg/mL

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C, 5.0 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- washed with DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg + 5 µL aqua dest.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL DPBS (Gibco, Cat No. 14040-91, Lot No.: 1737107)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL Sodium dodecyl sulfat (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3,
Lot No.: 40015277)
- Concentration (if solution): 5% in aqua dest

Duration of treatment / exposure:

15 min ± 0.5 miin

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3 tissues per dose group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three tissues

Value:

93

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

For detailed results see "Any other information on results" Table 1

Results of the  Pre-Experiments

The mixture of 10 mg test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixtures of 10 mg of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC_livingequalled 0%.

Results of the main experiment

Table 1: Results of the test item

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	0.742	0.816	0.676	0.069	0.086	0.086	0.620	0.724	0.736
	0.767	0.855	0.689	0.074	0.086	0.081	0.623	0.758	0.784
OD570(Blank-Corrected)	0.699	0.773	0.634	0.027	0.044	0.044	0.578	0.682	0.694
	0.724	0.813	0.647	0.031	0.044	0.039	0.581	0.716	0.742
Mean OD570Of The Duplicates (Blank-Corrected)	0.712	0.793	0.641	0.029	0.044	0.041	0.580	0.699	0.718
Total Mean OD570Of 3 Replicate Tissues (Blank-Corrected)	0.715*			0.038			0.665
SD OD570	0.076			0.008			0.075
Relative Tissue Viabilities [%]	99.5	110.9	89.6	4.1	6.1	5.8	81.0	97.7	100.4
Mean Relative Tissue Viability [%]	100.0			5.3**			93.0
SD Tissue Viability [%]^***	10.6			1.1			10.5
CV [% Viability]	10.6			20.6			11.3

^* Corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability.

^** Mean relative tissue viability of the three positive control tissues is 40%.

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

Table 2: Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean OD570 nmBlank	0.042	< 0.1	pass
Mean Absolute OD570 nmNK	0.757	0.6 ≤ NK ≤1.5	pass
Mean Relative Viability [%] PC	5.3	≤ 40%	pass
Max. SD of % Viability [%]	10.6	≤ 18%	pass

 

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In the present study the skin irritant potential of Chromium diboride (> 99.4 % purity) was analysed according to OECD 439 using the EPISKIN-Standard Model™ (EPISKIN-SM^TM), a reconstituted three-dimensional human epidermis model to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects (93.0 % mean relative tissue viability). As the relative mean tissue viability after 15 min of exposure and 42 h post-incubation was > 50%, the test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

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:

weight of evidence

Study period:

2017-01-17 to 2017-04-24

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:

adopted 02 October 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, 97633 Sulzfeld, Germany
- Age at study initiation: animal 1: approx. 44 weeks old; animal 2: approx. 41 weeks old
- Weight at study initiation: > 2 kg
- Housing: ABS-plastic or Noryl rabbit cages, floor 4200 cm², in an air-conditioned room
- Diet (e.g. ad libitum): ad libitum - Free access to autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Water (e.g. ad libitum): ad libitum - Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least 5 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 g of the test item was applied to the test site.

Duration of treatment / exposure:

single treatment

Observation period (in vivo):

The animals were observed for 72 hours after dosing.

Number of animals or in vitro replicates:

2 rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The treated eye was rinsed with physiological saline (0.9% NaCl) 1 hour after application.

SCORING SYSTEM: Grading system for ocular lesions according to OECD 405

TOOL USED TO ASSESS SCORE: To detect cornea lesions during the observation period, the treated eyes were examined with the aid of a fluorescein solution. The eyes were rinsed with physiological saline 0.9% NaCl after the examination.

TEST PROCEDURE:
Initial test: The in vivo test was performed initially using one animal.
Confirmatory test: The results of the initial test did not indicate the test item to be corrosive or a severe irritant to the eye using the procedure described. In order to confirm the response, one additional animal was treated in the same manner. According to OECD 405, section 23, treatment of a third animal was omitted as results from the second animal were sufficient to allow for hazard classification determination.

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

A single ocular application of the test item chromium diboride to rabbits at a dose of 0.1 g produced slight irritant effects, which were fully reversible within 48 hours. Upon fluorescein examinations at the end of the observation period of 72 hours no corneal lesions were found in any animal.

Other effects:

Neither mortalities nor significant clinical signs of toxicity but local effects were observed. Moreover, the body weight development of all animals was within the expected range

Table 1: Eye Irritation Scores - Animal No. 1
Observation	Animal No. 1, Eye Irritation Scores Post-Application After								Average Score (24, 48 and 72 hours)
	1 hour		24 hours		48 hours		72 hours
	Test item	Control	Test item	Control	Test item	Control	Test item	Control
Redness	1	0	1	0	0	0	0	0	0.33
Chemosis	0	0	0	0	0	0	0	0	0
Iris	0	0	0	0	0	0	0	0	0
Cornea	0	0	0	0	0	0	0	0	0

Table 2: Eye Irritation Scores - Animal No. 2
Observation	Animal No. 2, Eye Irritation Scores Post-Application After								Average Score (24, 48 and 72 hours)
	1 hour		24 hours		48 hours		72 hours
	Test item	Control	Test item	Control	Test item	Control	Test item	Control
Redness	1	0	1	0	0	0	0	0	0.33
Chemosis	0	0	0	0	0	0	0	0	0
Iris	0	0	0	0	0	0	0	0	0
Cornea	0	0	0	0	0	0	0	0	0

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vivo acute eye irritation/corrosion study in rabbits, chromium diboride was tested negative regarding irritation to the eye.

Executive summary:

In a primary eye irritation study according to OECD 405, 0.1 g of chromium diboride (>99.4 % purity) was applied into the conjunctival sac of one eye of two male New Zealand White Rabbits, Crl: KBL (NZW). The treated eye was rinsed with physiological saline 1 hour after the application. The untreated eye served as control. Control and treated animals were observed for a period of 72 hours.

Under the conditions of the present study, a single ocular application of the test item to rabbits at a dose of 0.1 g produced slight irritant but not corrosive effects in both animals, which were fully reversible within 48 hours in both animals. Neither mortalities nor significant clinical signs of toxicity were observed. Upon fluorescein examinations at the end of the observation period of 72 hours no corneal lesions were found in any animal.

On the basis of the test results and in conformity with the criteria given in Annex I of Regulation (EC) 1272/2008, the substance should be not classified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Chromium diboride (> 99.4% purity) was tested negative for acute dermal irritating properties in a study conducted according to OECD test guideline 439. In a screening study for identifying ocular corrosives and severe irritants (BCOP, OECD 437) no prediction could be made regarding the classification of Chromium diboride and further testing was required. Thus, an acute eye irritation/corrosion study in vivo (OECD 405) was conducted in rabbits. Only slight irritating effects (grade 1) were observed in both animals. These effects were fully reversible within 48 hours after application. Based on these results, Chromium diboride is considered to be not irritating to the skin and/or eye.

Justification for classification or non-classification

Chromium diboride (> 99.4% purity) was tested negative for acute dermal irritating and eye irritating/corrosive properties in studies conducted according to OECD 439 and OECD 405. Therefore, Chromium diboride does not warrant classification for skin and/or eye irritation.