isliFe

Endpoint summary

Description of key information

An in vitro skin irritation with reconstructed human epidermis (OECD 439) has been performed on the test substance.

In accordance with the Regulation EC No. 1272/2008, the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.

An in vitro isolated chicken eye test (OECD 438) has been performed on the test substance. In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item Acetic acid, oxo-, sodium salt, reaction products with

ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method.

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:

03 May 2019 - 06 June 2019

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no : TROD7BB06

Test system:

human skin model

Remarks:

Episkin SA

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

The 0.50 cm² reconstructed epidermises (Episkin SA, RHE/S/17 Batch No. 19-RHE-096) were received on 04 June 2019. The 4 additional killed Human skin model surfaces were defrozen the day of the treatment. 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 6 wells culture plate which had been previously filled with 300 μL of growth medium (Episkin SA, batch No. 19 SGM 065) during 2 hours and 40 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 (EpiskinSA, batch No. 19 SMM 022).

42 minutes after the test item application, the human epidermises were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 3630319). The rinsed tissues were checked for any coloration: after the rinse, the surface of the epidermises was slightly red with a darker outline. The 4 additional control tissues were darker.
The epidermises were incubated for a 41 hours and 40 minutes 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 responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal.
The skin samples were placed in 300 μL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. except for the 2 living and 2 killed Human skin model surfaces for the non-specific colour control which were placed in the maintenance medium (Episkin SA, batch No. 19 SMM 022).
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 microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied as supplied, to the epidermal surface of 3 living Reconstructed Human epidermis, at the dose of 16 mg per 0.50 cm² of human skin model, during 42 minutes at room temperature.

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 hours and 40 minutes

Number of replicates:

3 living Reconstructed Human Epidermis

Two killed Human skin tissues were treated in order to generate non-specific MTT reduction control and 2 additional killed and 2 additional living control tissues were treated to generate non-specific living and killed colour controls.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

86.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

mean tissue viability 1.3% for 5% sodium dodecy sulfate solution

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.
No hazard statement or signal word is required.

Executive summary:

In accordance with the Regulation EC No. 1272/2008, the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) 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

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 June 2019

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch TROD7BB06

Species:

chicken

Details on test animals or tissues and environmental conditions:

The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay. The age and weight of the chickens used in this test method are that of spring chickens traditionally
processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 03 June 2019 at 8:40 am. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg per eye

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

0, 30, 75, 120, 180 and 240 minutes (± 5 minutes)

Number of animals or in vitro replicates:

3

Details on study design:

Test item
Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, after being reduced in fine powder, during 10 seconds to the cornea 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. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

Controls
Concurrent negative control (physiological saline – Dutscher Batch No. 3013524) and positive control reduced in fine powder (sodium hydroxide – Fisher Scientific, Batch No. 1550248) were included in this experiment. One eye was treated with the negative control (under the same conditions as the eyes treated with the test item) and three eyes were treated with the positive control.

Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

METHODS FOR MEASURED ENDPOINTS:
All observations of the cornea and measurement of corneal thickness were performed using a Haag- Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 91⁄2, equalling 0.095 mm. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after test item exposure) were determined at 0, 30, 75, 120, 180 and 240 minutes (± 5 minutes) after the post treatment rinse.

SCORING SYSTEM:
- Mean corneal swelling (%): Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. It was expressed as a percentage and was calculated from corneal thickness measurements. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.
- Mean maximum opacity score: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.
- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.

Irritation parameter:

cornea opacity score

Run / experiment:

maximal mean score of 3 replicates

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class I

Irritation parameter:

fluorescein retention score

Run / experiment:

mean score

Value:

2.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class IV

Irritation parameter:

percent corneal swelling

Run / experiment:

maximal mean

Value:

8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: corresponding to ICE class II

Interpretation of results:

study cannot be used for classification

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.

Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

The test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) was applied, after being reduced in fine powder, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 438 adopted 09 October 2017 and the test method B.48 – Commission Regulation (EU) No. 1152/2010 dated 08 December 2010 (EU Journal L324) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.3, corresponding to ICE class I;

- mean score of fluorescein retention: 2.7, corresponding to ICE class IV;

- maximal mean corneal swelling: 8%, corresponding to ICE class II.

The combination of the three endpoints for the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) was 1 x IV, 1 x II, 1 x I.

The combination of the three 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 three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation/corrosion:

The substance is not classified a skin irritant because in the selected test it doesn't meet the classification criteria of the CLP regulation n. 1272/2008.

Eye irritation:

The substance is classified an eye irritant based on the ICE in vitro test (inconclusive for either serious eye damage and no irritation).