Dihydrogen hexahydroxyplatinate, compound with 2-aminoethanol (1:2)

Endpoint summary

Description of key information

In an in vitro EpiDerm skin irritation assay conducted in accordance with OECD guideline 439 and to GLP, dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2) was cytotoxic and, hence, irritant to skin (Spruth, 2016a).

 

In an in vitro EpiDerm skin corrosion assay conducted in accordance with OECD guideline 431 and to GLP, dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was corrosive to skin and classified as sub-category 1B-and-1C (Spruth, 2016b).

 

In an in vitro bovine corneal opacity and permeability (BCOP) assay conducted in accordance with OECD guideline 437, the In Vitro Irritancy Score (IVIS) for dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was calculated to be 4.585. No classification conclusion concerning irritant or corrosive potential of the test item can be made (Spruth, 2016c).

 

No relevant respiratory tract irritation data were identified.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 February - 26 April 2016

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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other:

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM (EPI-200-SCT, Lot no. 23312) 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 details below)

Details on animal used as source of test system:

Not applicable

Justification for test system used:

No data

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm is a three-dimensional reconstructed human epidermis model, comprised of normal, human-derived epidermal keratinocytes. Multiple layers of viable epithelial cells were present under a functional stratum corneum. The skin model also had a stromal component layer. Stratum corneum was multi-layered with the necessary lipid profile to produce a functional barrier with robustness to resist rapid penetration of cytotoxic markers.

Corrosive materials are identified by their ability to produce a decrease in cell viability (as determined, for example, by using the MTT reduction assay) below defined threshold levels at specified exposure periods. The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the underlying cell layers.

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:

- Amount(s) applied (volume or weight with unit): 50 μL of undiluted test item were applied to the skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface which was moistened with sterile deionised water to ensure adequate contact with the skin. Two replicate tissues for each treatment (exposure periods) were employed. At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL deionised water was added to each of the two negative control skin units.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL KOH was added to each of the two positive control skin units.
- Concentration (if solution): 8N solution

Duration of treatment / exposure:

3 minutes or 1 hour (37°C, 5% CO2 and 95% humidity)

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Score is a percentage of the negative control

Run / experiment:

mean (3 minute time point)

Value:

51.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The mean OD of the negative control of 2 tissues was 1.477 (3-minute exposure) 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 8N KOH was 7.0% (3-minute exposure) of the negative control and, hence well below the 15% cut-off value at the 1-h exposure.

Remarks on result:

other: possible indication of corrosivity

Remarks:

Mean relative viability <=50% the test substance is considered to be corrosive to skin (classified as sub-category 1A)

Irritation / corrosion parameter:

% tissue viability

Remarks:

Score is a percentage of the negative control

Run / experiment:

mean (1 hour time point)

Value:

12.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

The mean OD of the negative control of 2 tissues was 1.401 (1-hour exposure) 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 8N KOH was 9.6% (1-hour exposure) of the negative control and, hence well below the 15% cut-off value at the 1-h exposure.

Remarks on result:

other: positive indication of corrosivity

Remarks:

Mean relative viability >=50% (after 3 minutes) and <15% (after 1 hour) the test substance is considered to be corrosive to skin (classified as sub-category 1B-and-1C)

Other effects / acceptance of results:

The standard deviation of all replicates determined (at 20-100% viability) was below the limit of acceptance of 30%. Hence, all acceptance criteria were fulfilled.

No discolorations were noted in the test for colour change under aqueous conditions. Additionally, no change of colour was noted in the test for MTT interference potential. Therefore the test item did not interact with the MTT measurement and no additional test had to be performed.

 

The mean optical density (OD) of the negative control of 2 tissues was 1.477 (3-minute exposure) or 1.401 (1-hour exposure) and was well within the acceptable range of ≥ 0.8 to ≤ 2.8. The mean OD (and hence cell viability) of the positive control was 7.0% (3-minute exposure) or 9.6% (1-hour exposure) of the negative control and fulfilled the acceptance criterion of < 15% for the 1-hour exposure. The standard deviation of all replicates determined (at 20 - 100% viability) was below the limit of acceptance of 30%.Hence, all acceptance criteria required were fulfilled.

 

The summary of the results is given below:

	Optical density (OD) - 3 -minute exposure			OD - 1 -hour exposure
	OD (n=2 tissues)	SD	% OD540 compared to the control	OD (n=2 tissues)	SD	% OD540 compared to the control
Negative control deionised water	1.477	0.061	-	1.401	0.114	-
Dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2)	0.761	0.115	51.5	0.179	0.010	12.8
Positive control 8N KOH	0.103	0.022	7.0	0.134	0.004	9.6

Interpretation of results:

other: Sub-category 1B-and-1C since this test cannot resolve between the two

Conclusions:

In an in vitro EpiDerm assay conducted in accordance with OECD guideline 431 and to GLP, dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was corrosive to skin and classified as sub-category 1B-and-1C.

Executive summary:

Dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was tested for skin corrosivity potential in an in vitro EpiDerm assay conducted in accordance with OECD guideline 431 and to GLP.

Cell viability was quantitatively measured using the MTT reduction assay with optical density being expressed as a relative percentage of that of the negative control. The mean cell viability following exposure to the test substance was calculated to be greater than 50% (51.5% of the negative controls) after a 3-minute exposure and less than 15% (12.8% of the negative controls) after a 1-hour exposure, and it was therefore considered to be corrosive to skin.

Under the conditions of this assay, dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) did not meet the criteria for classification as corrosive category 1A under GHS classification criteria but would be classified as sub-category 1B-and-1C.

Based on the results of this study, the test item should be classified as corrosive to the skin (category 1B) according to EU CLP criteria (EC 1272/2008).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No relevant human irritation/corrosion data were identified.

 

Dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was tested for skin irritation potential in an in vitro reconstructed human epidermis model (EpiDerm assay) conducted in accordance with OECD guideline 439 and to GLP. EpiDerm is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum and irritant test materials are identified by their ability to decrease cell viability below defined threshold limits. Cell viability was quantitatively measured using the MTT reduction assay with optical density being expressed as a relative percentage of that of the negative control. If the resulting mean relative viability (as adjusted for intrinsic colour) is less than or equal to 50% of the negative control, the test substance is considered to be irritant to skin. The mean cell viability following 60-minute exposure to the test substance was calculated to be less than 50% (17.9% of the negative controls), and it was therefore considered to be cytotoxic and predicted to be irritant to skin. The positive and negative controls were considered valid. Under the conditions of this assay, dihydrogen hexahydroxyplatinate/2-aminoethanol (1:2) would be classified as "irritant" (Category 2) under GHS classification criteria (Spruth, 2016a). Since this test cannot resolve between GHS Categories 1 and 2, further information on skin corrosion is required to determine the final classification (see below).

 

Dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was tested for skin corrosivity potential in an in vitro EpiDerm assay conducted in accordance with OECD guideline 431 and to GLP. Cell viability was quantitatively measured using the MTT reduction assay with optical density being expressed as a relative percentage of that of the negative control. The mean cell viability following exposure to the test substance was calculated to be greater than 50% (51.5% of the negative controls) after a 3-minute exposure and less than 15% (12.8% of the negative controls) after a 1-hour exposure, and it was therefore considered to be corrosive to skin. Under the conditions of this assay, dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) did not meet the criteria for classification as corrosive category 1A under GHS classification criteria but would be classified as sub-category 1B-and-1C (Spruth, 2016b).

 

In an in vitro bovine corneal opacity and permeability assay conducted in accordance with OECD guideline 437 and to GLP, dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) was applied to isolated bovine corneas for 10 minutes, followed by an incubation period of 120 minutes. The IVIS calculated from individual scores for induced opacity (decreased light transmission through the cornea) and permeability (passage of sodium fluorescein dye through the cornea) was 4.585, which is above the cut-off value of 3 (no category) and below the cut-off value of 55 (identifying test substances as inducing serious eye damage). Hence, no classification conclusion concerning irritant or corrosive potential of the test item can be made (Spruth, 2016c). Substances that are corrosive to the skin are considered as leading to serious damage to the eyes. Consequently, no further testing is necessary and dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) should be classified for eye effects in Category 1 according to EU CLP criteria (EC 1272/2008).

 

No respiratory tract irritation data were identified. A new study was not conducted as it is not a REACH Standard Information Requirement.

Justification for classification or non-classification

Based on the results of the available in vitro skin corrosion study, dihydrogen hexahydroxyplatinate compound with 2-aminoethanol (1:2) should be classified as corrosive to the skin (category 1B) according to EU CLP criteria (EC 1272/2008).

According to ECHA guidance on the application of CLP criteria (ECHA, 2017b), “if a substance or mixture is classified as Skin corrosive Category 1 then serious damage to eyes is implicit…thus, the corrosive substance or mixture is also classified, but the corresponding hazard statement is not indicated on the label and there is no need to proceed with classification for eye effects”. HHPA:2AE is classified for skin effects as corrosive sub-category 1B. Consequently, the compound is classified for eye effects in Category 1 under EU CLP.