Dirhodium trioxide

Endpoint summary

Description of key information

Skin irritation potential of dirhodium trioxide is determined in a GLP complaint study according to OECD439. Dirhodium trioxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

Eye irritation potential of dirhodium trioxide is determined in a GLP compliant study according to OECD437. Dirhodium trioxide is not classified as severe irritant and is not corrosive according to UN GHS classification.

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:

28 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to GLP

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Reconstructed Human Epidermis Test method

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

purity: 100% Rh2O3x0.67 H2O or 95.5% Rh2O3 (based on Rh content of 77.4 wt%)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM (EPI-200, Lot no. 28611) MatTek In Vitro Life Science Laboratories, s.r.o, Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic

Source strain:

other: Reconstructed human epidermis model (see below)

Details on animal used as source of test system:

Not applicable

Justification for test system used:

The reconstructed human epidermis model system is suitable to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermal keratinocytes as cell source, representative tissue and cyto-architecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The procedure described under this test method allows the hazard identification of irritant substances in accordance with UN GHS Category 1 or Category 2.

EpiDerm is a three-dimensional reconstructed human epidermis model EpiDermTM, comprised of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability was measured by dehydrogenase conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue), into a blue formazan salt that was quantitatively measured after extraction from tissues. Irritant substances were identified by their ability to decrease cell viability below defined threshold levels (i.e.≤ 50% for UN GHS Category 1 or Category 2).

The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. MatTek determines the ET50 value following exposure to Triton X-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results.

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 of test item were applied to the skin model and uniformly covered the skin surface with an area of 0.63 cm2. The test item is a fine powder. For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline (D-PBS). Three tissue replicates were used.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL D-PBS was added to each of the three negative control skin units.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL SDS was added to each of the three positive control skin units.
- Concentration (if solution): 5% aqueous solution

Duration of treatment / exposure:

Exposure for 60 minutes: 35 minutes at 37°C, 5% CO2 and 95% relative humidity followed by 25 minutes at room temperature under sterile hood.

Duration of post-treatment incubation (if applicable):

The post-treatment incubation period of the rinsed tissues in fresh assay medium was 42 hours.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Score is a percentage of the negative control

Run / experiment:

mean (42 hour time point)

Value:

89.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The mean optical density (OD) of the negative control of 3 tissues was 1.870 and was well within the acceptable range of ≥ 0.8 to ≤ 2.8.

Positive controls validity:

valid

Remarks:

The viability of cells treated with the positive reference item was 6.3% of the negative control and fulfilled the acceptance criterion of <=20%.

Remarks on result:

no indication of irritation

Remarks:

Mean viability of cells exposed to dirhodium trioxide was 89.6% of the negative control and was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. Dirhodium trioxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

Other effects / acceptance of results:

Assay acceptability criteria:
1: The mean optical density (OD) of 3 negative control tissues was 1.870 and was well within the acceptable range of ≥0.8 to ≤2.8.
2: The viability of cells treated with the positive reference item, 5% SDS, was 6.3% of the negative control and fulfilled the acceptance criterion of ≤20%.
3: The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤ 18%.

The mean viability of cells exposed to dirhodium trioxide was 89.6% of the negative control and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of >50%. Dirhodium trioxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.870 and was well within the acceptable range of ≥0.8 to ≤2.8. The viability of cells treated with the positive reference item, 5% SDS, was 6.3% of the negative control and fulfilled the acceptance criterion of ≤20%.

The summary of the results is given below:

	Optical density (n = 3 tissues)	CV (%)	viability (%)		SD
Negative control (D-PBS)	1.870	4.9	100		4.9
Dirhodium trioxide	1.675	3.9	89.6		3.5
Positive control (5% SDS)	0.118	7.4	6.3		0.5

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vitro skin irritation test, using a reconstructed human epidermis model (EpiDermTM), dirhodium trioxide tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, did not show irritant properties and is therefore not classified as irritant (UN GHS no category).

Executive summary:

The cytotoxic properties of dirhodium trioxide to skin cells, which might lead to irritation of human skin, were determined by using an artificial three dimensional model of human skin (EpiDerm TM model). Three replicates were used for each treatment and concurrent control group. The optical density (OD) was determined by using the MTT reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

Dirhodium trioxide was applied as solid test item to the model skin surface, which was moistened with Dulbecco's phosphate buffered saline (D-PBS). D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SOS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

The mean viability of cells exposed to dirhodium trioxide was 89.6% of the negative control and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. Dirhodium trioxide was considered to be non-cytotoxic and predicted to be non-irritant to skin.

All acceptance criteria required were fulfilled and the study can be considered as valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 September 2018 - 6 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 09 October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EU) No. 2017/735 of 14 February 2017 amending Regulation (EC) No. 440/2008; EC method B.47: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

100% Rh2O3x0.67 H2O or 95.5% Rh2O3 (based on Rh content of 77.4 wt%)

Species:

other: Bovine eyes from cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source of eyes: Eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse. To minimize deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL.

Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.

Vehicle:

physiological saline

Remarks:

The test item was suspension suspended in a 0.9% sodium chloride solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

The test item was suspension suspended in a 0.9% sodium chloride solution with a final concentration of 20% (w/v). Dirhodium trioxide was thoroughly agitated on a whirl mixer and promptly administered to the cornea.

750 µL of the test or control items were added to completely cover the cornea’s epithelium in the anterior chamber.

Duration of treatment / exposure:

240 minutes (recommended exposure time for non-surfactant solids)

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

Not applicable

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: Only corneas from eyes free of defects were used. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded. The corneas were dissected with a 2 to 3 mm rim of sclera and mounted in corneal holders with anterior (epithelium) and posterior (endothelium) chambers.Beginning with the posterior chambers, the chambers were filled to excess with pre-warmed Eagle’s Minimum Essential Medium (EMEM) , while preventing bubble formation. The corneal holder was equilibrated at 32 °C ± 1 °C for at least one hour.

QUALITY CHECK OF THE ISOLATED CORNEAS: After the equilibration period, fresh pre-warmed EMEM was added to both chambers and baseline opacity readings were taken for each cornea. Corneas exhibiting macroscopic tissue damage (e.g. scratches, pigmentation, neovascularisation) or an opacity >7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated by use of an opacitometer. A minimum of three corneaswith opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment, solvent and positive control groups (three corneas/group).

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED: 20% Imidazole (CAS no. 288-32-4) in 0.9% sodium chloride solution

TREATMENT METHOD: open-chamber method, 240 minutes exposure period

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the exposure period the epithelium was washed with EMEM containing phenol red at least three times. Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible. The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber. After rinsing, the glass window was replaced on the anterior chamber to recreate a closed system and the chamber was filled with EMEM without phenol red.
- POST-EXPOSURE INCUBATION: not applicable

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer resulting in opacity values measured on a continuous scale.
- Corneal permeability: 1 mL sodium fluorescein solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface) was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32 ± 1 °C for 90 ± 5 minutes. The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader. Measurements at 490 nm were recorded as optical density (OD490). The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer using a standard 1 cm path length.
- Others (e.g, pertinent visual observations, histopathology): Corneal injury was assessed by evaluating the opacity and permeability of the cornea.

SCORING SYSTEM: After correcting the opacity and mean permeability (OD490) values for background opacity and the negative control permeability OD490 values, the mean opacity, and permeability OD490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The opacity and permeability values were also evaluated independently to determine whether the test item induced corrosivity or severe irritation through only one of the two endpoints.

DECISION CRITERIA: The IVIS cut-off values for identifying test chemicals as inducing serious damage (UN GHS Category 1) and test chemicals not requiring classification for irritation or serious eye damage (UN GHS No Category) are given hereafter.

IVIS UN GHS
≤ 3 No Category
>3 and ≤55 No prediction can be made
> 55 Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

1.814

Vehicle controls validity:

valid

Remarks:

IVIS score 0.9% NaCl = 0.806 (SD = 0.491)

Positive controls validity:

valid

Remarks:

IVIS score 20% Imidazol = 97.538 (SD = 5.300)

Remarks on result:

no indication of irritation

Remarks:

The calculated IVIS of 1.814 ± 1.314 is below the cut-off value of 3 (UN GHS no category) and consequently the test item is not classified as a severe irritant and is not corrosive according to UN GHS classification.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: see Table 1

The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of 0.491±0.310and a mean permeability value of 0.021 ± 0.012. The calculated IVIS value of0.806±0.491was well below the cut-off value of 3 (UN GHS no category).

The corneas treated with the positive control item 20% Imidazole in 0.9% NaCl solution revealed a mean opacity value of68.898±5.730and a mean permeability value of 1.909 ± 0.059 compared to the solvent control. The calculated IVIS value of97.538±5.300was within two standard deviations of the current historical mean and well above the cut-off value of 55. Hence, the acceptance criteria for the test were fulfilled.

Following treatment withDirhodium trioxide,amean opacity of1.674±1.243and a mean permeability value of 0.009 ± 0.005 compared to the negative control were determined. The calculated IVIS of1.814±1.314isbelow the cut-off value of 3 (UN GHS no category)andconsequently the test item is not classified as a severe irritant and is not corrosive according to UN GHS classification.Furthermore, it can be concluded that the test item Dirhodium trioxiderequiresnoclassification for eye irritation or serious eye damage under the UN GHS classification system.

The IVIS values are given in the table below:

 

	Cornea No.	Opacity	Permeability	IVIS
				Per Cornea	Per Group
					Mean	SD
0.9% NaCl	1	0.239	0.010	0.389	0.806	0.491
	2	0.837	0.034	1.347
	3	0.398	0.019	0.683
20% Dirhodium trioxide	4	0.306	0.004	0.366	1.814	1.314
	5	2.736	0.013	2.931
	6	1.979	0.011	2.144
20% Imidazol	7	62.418	1.957	91.773	97.538	5.300
	8	73.294	1.927	102.199
	9	70.983	1.844	98.643

SD: standard deviation

OD: optical density

Interpretation of results:

GHS criteria not met

Conclusions:

Dirhodium trioxide tested in the in vitro BCOP test method, had an IVIS value of 1.814, which is below the cut-off value of 3 (UN GHS no category) and consequently the test item is not classified as a severe irritant and is not corrosive according to UN GHS classification. It can be concluded that the test item Dirhodium trioxide requires no classification for eye irritation or serious eye damage under the UN GHS classification system.

Executive summary:

The eye irritation potential of Dirhodium trioxide was determined in an in vitro system (Bovine Corneal Opacity and Permeability Assay (BCOP) test method) accodring to OECD test guideline 437. The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% Dirhodium trioxide. 0.9% NaCl solution was used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item.

The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the controls was 240 minutes. The optical density (OD) was measured at a wavelength of 490 nm.

The acceptance criteria of validity were fulfilled in this test.

Following treatment with Dirhodium trioxide a mean opacity of 1.674±1.243 and a mean permeability value of 0.009 ± 0.005 compared to the negative control were determined. The calculated IVIS of1.814±1.314 is below the cut-off value of 3 (UN GHS no category) and consequently the test item is not classified as a severe irritant and is not corrosive according to UN GHS classification.It can be concluded that the test item Dirhodium trioxide requires no classification for eye irritation or serious eye damage under the UN GHS classification system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Dirhodium trioxide requires no classification for skin irritation / corrosion and eye irritation or serious eye damage under the CLP / UN GHS classification system.