Chromium tris((2-ethylhexanoate)

Endpoint summary

Description of key information

Skin irritation: Not Irritating to the skin.

Eye irritation: Not irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-10 to 2017-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

dated 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Test method B.40bis Council regulation n°440/2008

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 23 October 2015

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

The 0.6 cm² reconstructed epidermis (epiCS, Cell Systems - batch n° 100-AF2446-1) were received on 17 January 2017. The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The insert was placed in a 6 wells culture plate which had been previously filled with 1 mL of culture medium (Cell Systems batch n° 305-AG0224). The culture dishes were incubated at 37 +/- 2°C, 5% CO2 during 20 hours and 45 minutes before treatment. Just before the treatment, the culture medium was replaced by a new culture medium (Cell Systems, batch n° 305-AG0224).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The test item was applied, as supplied,after being applied on a nylon mesh, at the dose of 25 mg,during 3 minutes and during 1 hour at room temperature, to the epidermal surface of 4 living human skin models, including two for the non-specific colour (NSC) control.
In the same experimental conditions, a positive control (8N KOH - Sigma batch n° SLB3295V) and a negative control (DPBS - PAN BIOTECH GmbH, batch 9510916) were carried out.

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

4 living human skin models (2 living for non-specific colour living control)

Details on study design:

3 minutes and 1 hour after the test item applicationn, the human epidermis was washed 20 times with 1 mL of DPBS (PAN BIOTECH GmbH, batch n° 9510916). The rinsed tissues were checked for coloration and presented green spots.
The cell viability was quatified by measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazol blue; CAS n° 298-93-1] reduction into blue formazan crystal. The skin sample was placed in MTT solution of 1 mg/L concentration, except for the two tissues for the non-specific control which were placed in MTT assay medium (Cell Systems batch n° 303-AG0224), for 3 hours between 36.8°C and 37.8°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extraction in isopropanol (1:3).
The absorbance was measured in triplicate of MTT extract.
The measured absorbances were proportional to the number of living cells.

The measurement of OD was performed using the Elx800 absorbance microplate reader (controlled and calibrated every year if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes after the test item application

Value:

78.19

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour after the test item application

Value:

84.55

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The results are expressed as a viability percentage compared with the negative control:
viability % = OD test item / OD negative control x 100
Because the test item was potentially causing colour interferences, true viability was calculated as follows:
true viability % = [(OD of living tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 100

The mean viability of epidermis skins treated with the positive control item (potassium hydroxide 8N) was 58.81% and 0.74% respectively 3 minutes and 1 hour after the application.

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with Regulation EC 1272/2008, the results obtained under these experimental conditions unable to conclude that the test item does not have to be classified in category 1 'Corrosive'.
The hazard statement 'H314: Causes severe skin burns and eye damage' with the signal word 'Danger' are not required.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-01 to 2017-06-09

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:

adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 27 April 2017

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

The 0.5 cm² reconstructed epidermis (Episkin SA, RHE/S/17 - batch n° 17-RHE-035) were received on 21 March 2017. The same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in a 6 wells culture plate which had been previously filled with 1 mL of growth medium (EpiSkin SA, batch n° 17 MPE 022) during 2 hours and 35 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate whic had been previously filled with 300 µL of maintenance medium (EpiSkin, batch n° 17 MA 018).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The test item was applied at an approximate dose of 16 mg to the entire surface of the epidermal, previously moistened with 10 µL of distilled water, of 5 living skin models, including two for the non-specific colour (NSC) control, during 42 minutes at room temperature.
In the same experimental conditions, a positive control (5% SDS), and a negative control ( DPBS - VWR - batch n° 6MB235) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA - batch n° STBF1623V) in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment.
To ensure good contact with the epidermis, during all the treatment period, all liquid items were recovered with a nylon mesh provided by Episkin SA.

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 hours and 35 minutes

Number of replicates:

5 living skin models (including 2 living for non-specific colour living control)

Vehicle:

unchanged (no vehicle)

Details on study design:

42 minutes after the test item application, the nylon mesh was removed and the human epidermis were washed with 25 x 1 mL of DPBS (VWR, batch n° 6MB235). The rinsed tissues were checked for any coloration and noted to be green because of residue of the test item (parameter identified and managed with the additon of the non-specific colour control to the study).

They were incubated for 41 hours and 35 minutes post-treatment incubation-period in fresh medium at 37°C, 5% CO2. Then, the epidermis were put in contact with the MTT solution.

The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazol blue; CAS n° 298-93-1)] reduction into blue formazan crystal. The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/L, except for the two for non-specific colour control which were placed in the maintenance medium (Episkin SA, batch n° 16 MA 018) for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol.
The OD was measured in triplicate of MTT extract.
The measured OD are proportional to the number of living cells.

The measurement of OD was performed using the ELx800 absorbance microplte reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Irritation / corrosion parameter:

% tissue viability

Value:

78

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean viability (%) = 1.4 (5% Sodium dodecyl sulfate)

Other effects / acceptance of results:

The results were expressed as a viability percentage compared with the negative control:
% viability = ODtest item / OD negative control * 100

Because the test item was identified as potentially causing colour interferences, the true viability was calculated as follows:
% true viability = [(OD of living tissues exposed to the test item - OD of living tissues exposed to the test item incubated with medium without MTT) / OD living tissues exposed to negative control] * 100

Results after treatment with test item and the controls

Dose group	Treatment	Mean OD Skin 1*	Mean OD Skin 2*	Mean OD Skin 3*	Mean OD Product	Mean Viability %
Negative control	42 min	0.768	0.872	0963	0.868	100.0
Positive control	42 min	0.012	0.011	0.014	0.012	1.4
Test item	42 min	0.721	0.752	0.810	0.761	87.7
Test item NSC Control	42 min	0.099	0.069		0.084	9.7
Test item corrected						78.0

OD = Optical Density

The standard deviations between the % variabilities of the test item, the positive and negative controls were respectively 21.7 - 0.1 - 0.5%. The threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method” is 18%. Considering the results obtained, this deviation is considered as without impact on the conclusion of the study.

EVALUATION AND INTERPRETATION OF THE RESULTS

The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarely set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:

- The test item is considered to be non irritant to the skin if the viability after 42 minutes of exposure and  42 hours of post-treatment incubation is > 50%.

- The test item is considered to be irritant to the skin if the viability after 42 minutes of exposure and  42 hours of post-treatment incubation is  ≤ 50%. In accordance with the REgulation EC n° 1272/2008, the test item is to be classified in Category 2 'Irritant'. The corresponding hazard statement is 'H315: Causes skin irritation' with the signal word 'Warning'.

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation EC n° 1272/2008, the test item has to be considered as non irritant to the skin in accordance with UN GHS No category. No hazard statement or signal word are required.

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:

2017-02-28 to 2017-06-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dated 27 April 2017

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

The test item was applied, on three gauzes (0.481g, 0.474g, and 0.508g) in order to cover the entire surface of the cornea. The gauzes were subsequently applied to 3 unucleated chicken eyes during 10 seconds such that the entire surface of the cornea was evenly covered with the test item. Then the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.
Concurrent negative control (physiological saline - Dutscher batch n° 3012316) and positive control (sodium hydroxide - Sigma batch n° MKBP7805V) were included in this experiment. One eye was treated with the negative control and three eyes were treated with the positive control.

Duration of post- treatment incubation (in vitro):

All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device n° I. For the measurement of corneal thickness, the slit-width was set at 9½ equalling 0.095 mm.
Treated corneas are evaluated pretreatment and starting at 30, 75, 120, 180 and 240 minutes (+/- 5 min.) after the post-treatment rinse.

Number of animals or in vitro replicates:

3 chicken eyes for main test, 1 chicken eye for negative control and 3 chicken eyes for positive control

Irritation parameter:

cornea opacity score

Remarks:

mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 0 min

Irritation parameter:

cornea opacity score

Remarks:

mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 30 min

Irritation parameter:

cornea opacity score

Remarks:

mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 75 min

Irritation parameter:

cornea opacity score

Remarks:

mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 120 min

Irritation parameter:

cornea opacity score

Remarks:

mean

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 180 min

Irritation parameter:

cornea opacity score

Remarks:

mean

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 240 min

Irritation parameter:

fluorescein retention score

Remarks:

mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 0 min

Irritation parameter:

fluorescein retention score

Remarks:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 30 min

Irritation parameter:

percent corneal swelling

Remarks:

mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 30 min

Irritation parameter:

percent corneal swelling

Remarks:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 75 min

Irritation parameter:

percent corneal swelling

Remarks:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 120 min

Irritation parameter:

percent corneal swelling

Remarks:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 180 min

Irritation parameter:

percent corneal swelling

Remarks:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: time = 240 min

Other effects / acceptance of results:

Test item - Chromium 2-ethylhexanoate:
Corneal opacity: maximal mean score = 0.2 at 240 min corresponding to ICE class I
Fluorescein retention: maximal mean score = 1.0 at 30 min corresponding to ICE class II
Corneal swelling: maximal mean = + 1% at 240 min corresponding to ICE class I
Combination of the 3 endpoints: 2 x I, 1 x II

Positive control:
The combination of the 3 endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore the positive control is classified as 'Corrosive/severe irritant' as expected.

Negative control:
The combination of the 3 endpoints for the negative control, physiological saline, was 3 x I. Therefore the negative control is classified as 'No category' as expected.

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with Regulation EC 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No Category). No signal word and hazard statement are required.

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

Skin corrosion:

The in vitro skin corrosion test (Colas, 2017) conducted according to OECD guideline 431 is considered as the key study for skin corrosion and will be used for classification. 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 78.19% and 84.55%, versus 58.81% and 0.74% respectively with the positive control item (Potassium hydroxide 8N).

Therefore, the classification criteria according to regulation EC 1272/2008 as corrosive to the skin are not met, hence no classification is required.

Skin irritation:

Reference Floriot (2017) is considered as the key study for skin irritation and will be used for classification. According to the OECD 439 guideline, the mean corrected percent viability of the treated tissues was 78.0%, versus 1.4% in the positive control (5% Sodium dodecyl sulfate) after 42 minutes of exposure and 42 hours of post-treatment incubation.

Results do not meet the criteria for classification (tissue viability after 42 minutes of exposure and 42 hours post-treatment incubation > 50%) according to regulation (EC) 1272/2008 as skin irritant, hence no classification is required.

Eye irritation:

The reference Floriot (2017) is considered as the key study for severe eye irritation and will be used for classification.

According to the Isolated chicken eye test method, the ocular reactions observed were:

- maximal mean score of corneal opacity: 0.2 corresponding to ICE class I;

- maximal mean score of fluorescein retention: 1.0, corresponding to ICE class II;

- maximal mean corneal swelling: 1%, corresponding to ICE class I.

Therefore, the classification criteria according to regulation EC 1272/2008 are not met, hence no classification is required.

Respiratory irritation:

The available data do not allow any conclusion on respiratory irritation.