Salt reaction of cobalt(2+) and C2/C8/C20 carboxylates

Endpoint summary

Description of key information

Skin irritation: not irritating to the skin

Eye irritation: not irritating 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:

2021-09-21 to 2021-12-20

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 14 June 2021

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in-vitro study there is no information on test animals.

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied after being reduced to fine powder, at the dose of 16 mg, during 42 minutes at room temperature, to the epidermal surface of 5 living rhCE tissue replicates (including 2 for non-specific colour living control) and 4 killed RhCE tissues replicates (including 2 fo the non-specific MTT reduction control and 2 for the non-specific colour killed control). The Reconstructed Human epidermis had been previously moistened with 10 µL of distilled water.
Under the same experimental conditions, a positive control (16 µL 5% sodium dodecyl sulfate (SDS)) and a negative control (16 µL of distilled water - ADL Prochilab - batch n° 201117) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA - batch n° STBJ3028) in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment to obtain a colourless solution. To ensure good contact with the epidermis, during all the treatment period, the control items were recovered with a nylon mesh provided by Episkin SA.

Duration of treatment / exposure:

42 minutes

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

HUMAN SKIN MODEL
The 0.50 cm2 reconstructed epidermis (Episkin SA, RHE/S/17 batch n°21-RHE-169) were received on 26 October 2021. The 4 additional killed Human skin model surfaces (Episkin SA, RHE/S/17 - batch n°21-RHE-057 frozen on 20 April 2021) were defrozen the same day.
The inserts (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of growth medium (Episkin SA, batch n° 21 SGM 118) fot 2 hours and 52 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA, batch 21 SMM 042).

EVALUATION OF DIRECT INTERACTION WITH MTT
The direct interaction of MTT with the test item was checked by adding 16 mg of the test item to 300 µL of solution of MTT at 1 mg/L. A yellow solution with purple test item was observed after 3 hours of incubation between 37.2°C and 37.4°C, 5% CO2. > Therefore, due to its colour, the test item was identified as porducing interference with the MTT reduction reading. Two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non-specific MTT reguction (NSMTT) control.

SPECTRAL ANALYSIS OF THE TEST ITEM IN ISOPROPANOL
The spectral properties at 570 nm of test item in isopropanol ware checked by adding 16 mg of the test item to 1.5 mL of isopropanol (same conditions as in the main test). A purple solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.446 which is higher than 0.08 (value corresponding to approximately twice the OD of the extracting solvent. > Therefore, the test item was identified as causing colour interference with the viability assay. Two viable control tissues were added to the study which underwent the entire testing procedure but were incubated in culture medium instead of MTT solution during the MTT incubbation step to generate a non-specific colour (NSC living) control.

> Therefore, as the test item was identified as interfering with MTT readin reduction and colour interference, two killed control tissues (NSC killed control) were added to the study, which underwent the entire testing procedure but were incubated in cluture medium instead of MTT solution during the incubation step.

GRADING OF REACTIONS
42 minutes after the test item application, the nylon mesh was removed and the human epidermies were washed with 25 x 1 mL of DPBS (Dutscher, batch n° 7380521). The rinsed tissues were checked for any coloration and noted to be white, comparable coloration to that of the negative control tissues.
The epidermises were incubated for 41 hours post-treatment incubation-period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.

The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme was responsibel 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 that is quantatively measured by Optical Density (OD) after extraction from tissues.
The measured OD are proportional to the number of living cells.

The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/L for 3 hours at 37°C, 5%CO2; except for the NSC living and killed controls which were placed in the maintenance medium (Episkin SA, batch n° 21 SGM 118) instead of MTT solution.

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 measurement of OD at 570 nm of formazan extracts was performed in triplicate samples 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.

TREATMENT OF THE RESULTS
The results were expressed as a viability percentage compared with the negative control:
% viability = OD test item / OD negative control * 100

As the test item is identified as producing both direct MTT reduction and colour interference, the true viability was calculated as follows:
% true viability = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item (NSMTT) - OD of living tissues exposed to the test item incubated with medium without MTT (NSC living) + OD of killed tissues exposed to test item incubated with medium without MTT (NSC killed))/OD living tissues exposed to negative control] * 100

Results for test items producing non specifc MTT reduction (%NSMTT) and/or non-specific colour (%NSC living) ≥ 50% of the negative control should be taken with caution.

For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, should be reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Value:

104.9

Negative controls validity:

valid

Remarks:

mean viability (%) = 100

Positive controls validity:

valid

Remarks:

mean viability (%) = 1.6

Remarks on result:

other:

Remarks:

Time point: after 42 min of exposure and 42 hours post-treatment incubation.

Other effects / acceptance of results:

- SD value of the % viability ≤ 18%
- Negative control: OD values of the 3 replicates in the range of ≥0.8 and ≤ 3.0.
- The optical density was measured after 1:2 dilution of the formazan extracts in isopropanol; the acceptability criterai should be in the range of ≥0.4 and ≤ 1.5.
- Positive control: mean viability < 40%.

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	0.949	0.870	0.791	0.870	100.0
Positive control	42 min	0.016	0.013	0.013	0.014	1.6
Test item	42 min	0.958	0.892	0.910	0.920	105.7
Test item NSMTT	42 min	0.008	0.010		0.009	1.0
Test item NSC killed	42 min	0.005	0.002		0.004	0.4
Test item NSC living	42 min	0.003	0.001		0.002	0.2
Test item corrected						104.9

OD = Optical Density

The standard deviations between the % variabilities of the test item, the positive and negative controls were respectively 3.9 - 0.2 - 9.1%. 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%.

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 skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-09-22 to 2021-11-18

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Vehicle:

unchanged (no vehicle)

Duration of treatment / exposure:

30 mg of the test item was applied, after being reduced to fine powder, and under fume hood, for 10 seconds to the cornea such that the entire surface of the cornea was evently covered with the test item. Then the eyes were rinsed twice wiith 10 mL of physiological saline at ambient temperature.
Concurrent negative control (physiological saline - Dutscher batch n° C0543A01) and positive control (sodium hydroxide - Fisher Scientific - batch n° 00000) were included in this experiment. One eye was treated with 30 µL of negative control and three eyes were treated with 30 mg of positive control. Then, the controls underwent the entire testing procedure.

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

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 = 45 min pre-treatment

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:

0.3

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:

0.5

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:

0.5

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:

0.5

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 = 45 min pre-treatment

Irritation parameter:

fluorescein retention score

Remarks:

mean

Value:

2

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:

11

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:

11

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:

13

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:

13

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:

13

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:

The ocular reactiond observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 0.5, corresponding to ICE class I;
- mean score of fluorescein retention: 2.0, corresponding to ICE class III;
- maximal mean corneal swelling: 13%, corresponding to ICE class II.
The combination of the three endpoints for the test item was 1 x I, 1 x III, 1 x II.

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.

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:

other: No prediction can be made.

Conclusions:

In accordance with Regulation EC 1272/2008, the results obtained under these experimental conditions lead to the category 'No prediction can be made' as defined by the O.E.C.D. test guideline n° 438. Therefore, the test item is not predicted as causing sever eye damage (Caetegory) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing is required to establish a definitive classification.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-10-04 to 2022-01-xx

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 18 June 2019

GLP compliance:

yes (incl. QA statement)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg

Duration of treatment / exposure:

Pre-treatment:
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 7380521). The tissues were incubated at standard culture conditions for 30 minutes.
Treatment:
The test item was applied after being reduced to fine powder, for 6 hours at standard culture conditions, at the dose of 50 mg to the entire surface of 4 living RhCE tissue replicates (including 2 for NSC living control) and 4 killed RhCE tissue replicates (2 for the NSMTT control and 2 for the NSC killed control).
In the same experimental conditions, a positive control (Methyl acetate - Sigma-Aldrich, batch No. BCBX8836) and a negative control (distilled water - ADL Prochilab - Batch No. 201117) were carried out. The controls were applied, as supplied, at the dose of 50 μL, to the surface of 2 RhCE tissue replicates during 6 hours at standard culture conditions.

Duration of post- treatment incubation (in vitro):

Post-exposure incubation period:
After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 7380521). The rinsed tissues were checked for any coloration and noted to be white, comparable coloration to that of the negative control tissues.
This rinsing step was followed by a 25-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue.
The RhCE constructs were then incubated for a 17 hours and 52 minutes post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

Number of animals or in vitro replicates:

2

Details on study design:

Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
The RhCE constructs were placed in 300 μL of a MTT solution at 1.0 mg/mL for 2 hours and 52 minutes at standard culture conditions. The NSC living and NSC killed control tissues were incubated in assay medium (MatTek Corporation, batch No. 111521ISA) instead of MTT solution in order to generate NSC controls.
The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol for 2 hours and 20 minutes at room temperature in the dark.
The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2).
The OD at 570 nm was measured in triplicate samples of formazan extracts.
The measured OD are proportional to the number of living cells.
The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Irritation parameter:

other: mean percent tissue viability  (migrated information)

Remarks:

mean

Value:

83.25

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: mean optical density

Value:

0.766

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptability criteria:
- Tissues treated with the positive control substance should show a mean tissue viability < 50%.
- The difference of viability between two tissue replicates should be less than 20%.
- Negative control: OD values of the two replicates should be in the range > 0.8 and < 2.5.
As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.25 for the negative control.

The results were expressed as a viability percentage compared with the negative control:

Viability % = Mean OD test item x 100/Mean OD negative control

As the test item was identified as producing both colour interference and direct MTT reduction, true tissue viability is calculated as follows:

True viability % = (Mean OD test item – Mean OD NSMTT– Mean OD NSC living + Mean OD NSC killed)x 100/

Mean OD negative control

NSMTT: killed tissues exposed to the test item

NSC living: living tissues exposed to the test item then incubated in medium instead MTT

NSC killed: killed tissues exposed to the test item then incubated in medium instead MTT

Evaluation and interpretation of the results

The OD values obtained with the replicate tissue extracts for each test item were used to calculate the mean percent tissue viability normalized to the negative control, which was set to 100%. The percentage tissue viability cut-off value distinguishing classified from non-classified test items is 60%. Results are interpreted as follows:

The test item is identified as not requiring classification and labelling according to UN GHS No Category:

➢ if the mean percent tissue viability after exposure and post-exposure incubation is > 60%.

In this case, no further testing in other test methods is required.

The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1):

➢ if the mean percent tissue viability after exposure and post-exposure incubation is ≤ 60%.

When the final mean percent tissue viability is ≤ 60%, further testing with other test methods will be required because the RhCE test method shows a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

The mean corrected percent tissue viability of the RhCE replicates treated with the test item was 83.25%, versus 29.83% in the positive control (Methyl acetate).

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions and in accordance with the Regulation EC No. 1272/2008, it is possible to conclude that the test item does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.
No hazard statement and the signal word are required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Justification for selection of skin irritation / corrosion endpoint:
Key study.

Justification for selection of eye irritation endpoint:
Key study.

Justification for classification or non-classification

Skin irritation

Reference Chrifi (2021) conducted according to OECD guideline 439 is considered as the key study for skin irritation and will be used for classification. The mean corrected percent viability of the treated tissues was 104.9% after 42 minutes of exposure and 42 hours of post-treatment incubation.

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

Eye irritation

The reference Chrifi (2021) conducted according to OECD 438 guideline is considered as the key study for severe eye damage.

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

- maximal mean score of corneal opacity: 0.5, corresponding to ICE class I;

- mean score of fluorescein retention: 2.0, corresponding to ICE class III;

- maximal mean corneal swelling: 13%, corresponding to ICE class II.

In accordance with regulation EC 1272/2008, the results obtained under these experimental conditions lead to the category 'no prediction can be made'. Additional testing was required to establish a definitive classification.

Reference Chrifi (2022) conducted according to OECD guideline 492 is considered as the key study for in-vitro eye irritation and will be used for classification.

The mean corrected percent tissue viability of the RhCE replicates treated with the substance was 83.25%, versus 29.83% in the positive control (methyl acetate).

In accordance with the regulation EC No. 1272/2008, it is possible to conclude that the substance does not require classification for eye irritation or serious eye damage.