Reaction products of “Fatty Acids, C16-18 and C18-unsatd., Me esters; sulfuric acid and sodium hydroxide

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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: 2018060002
pH: 7.60
moisture content: 30.29%

Test system:

human skin model

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. 20-RHE-011) were received on 21 January 2020. 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. 20 SGM 008) during 2 hours. 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 No. 20 SMM 003).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

16 μL

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours and 45 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

5.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

other: Category 1 or 2

Conclusions:

In accordance with the Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” or “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

Executive summary:

The aim was to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model). The test item was applied as supplied, at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 42 hours and 45 minutes post-incubation period at 37°C, 5% CO2.

Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 439 adopted 18 June 2019.

The mean corrected percent viability of the treated tissues was 5.7%, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation EC No. 1272/2008 and in absence of information on skin corrosion, the test item has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” or “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: 2018060002
pH: 7.60
moisture content: 30.29%

Test system:

human skin model

Remarks:

epiCS, Cell Systems

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

The 0.6 cm² reconstituted epidermis (epiCS, Cell Systems – Batch No.100-AJ0456-1) were received on 24 March 2020. 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 No. 305-AJ0638).
The culture plates were incubated at 37±2°C, 5% CO2 during 22 hours and 15 minutes before treatment. Just before the treatment, the culture medium was replaced by a new culture medium (Cell Systems, Batch No. 305-AJ0638).

Vehicle:

unchanged (no vehicle)

Details on test system:

The test item was applied as supplied at the dose of 50 μL to the epidermal surface of the 2 living human skin models during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL

Duration of treatment / exposure:

observations at 3 min and 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 3 minutes

Value:

90.99

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 1 hour

Value:

93.96

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

other: not category 1 (corrosive)

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item RICOIL EN 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.

Executive summary:

The aim of the study was to evaluate the possible corrosive effects of the test item RICOIL EN after topical administration on in vitro human reconstituted epidermis (epiCS®, supplied by CellSystems®).

The test item RICOIL EN was applied as supplied, at the dose of 50 μL, to 2 living Human skin model surfaces (epiCS®, supplied by CellSystems®) during 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 431 dated 18 June 2019.

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 90.99% and 93.96%, versus 17.60% and 1.22%, respectively, with the positive control item (potassium hydroxide 8N).

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item RICOIL EN 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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

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: 2018060002
pH: 7.60
moisture content: 30.29%

Species:

chicken

Details on test animals or tissues and environmental conditions:

The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820Etauliers, 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 13 January 2020 at 8:28 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.

The eyes were enucleated at Phycher on 13 January 2020 at 10:00 am

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 μL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

Number of animals or in vitro replicates:

test item : 3 eyes
postive control : 3 eyes
negative control : 1 eye

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

mean

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

A histopathological examination was performed. The study meets the acceptance criteria as the negative control item and the positive control item fall within the Not Classified and Severe irritant classification, respectively. Following the application of the test item RICOIL EN, there was very slight (1/2) erosion of the
superficial part of the superficial corneal epithelium in 1 out 3 eyes examined. According to these findings, the test item was classified as negative for GHS Category 1 for non-extreme pH (2 < pH < 11.5) detergents and surfactants.

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation (EC) No. 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.
However, these results are in contradiction with the results obtained with skin irritation in vitro (OECD 439). Indeed, the product has been classified in category 1 "Corrosive" or category 2 "Irritant" for its effects on the skin.
A histopathological examination was performed. The study meets the acceptance criteria as the negative control item and the positive control item fall within the Not Classified and Severe irritant classification, respectively. Following the application of the test item RICOIL EN, there was very slight (1/2) erosion of the superficial part of the superficial corneal epithelium in 1 out 3 eyes examined. According to these findings, the test item was classified as negative for GHS Category 1 for non-extreme pH (2 < pH < 11.5) detergents and surfactants.
Therefore, in accordance with the Regulation (EC) No. 1272/2008, the test item RICOIL EN is classified in the category “no prediction can be made”. It 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.
As a conservative approach and in the absence of additional testing (in vitro and/or in vivo), the substance is classified as irritant to eye (category 2).

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 was applied, as supplied, at the dose of 30 μL, 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 25 June 2018.

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: 0.8, corresponding to ICE class II;

- maximal mean corneal swelling: 4%, corresponding to ICE class I.

The combination of the three endpoints for the test item was 2 x I, 1 x II.

The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, 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 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.

However, these results are in contradiction with the results obtained with skin irritation in vitro (OECD 439).

Indeed, the product has been classified in category 1 "Corrosive" or category 2 "Irritant" for its effects on the skin.

A histopathological examination was performed. The study meets the acceptance criteria as the negative control item and the positive control item fall within the Not Classified and Severe irritant classification, respectively. Following the application of the test item RICOIL EN, there was very slight (1/2) erosion of the superficial part of the superficial corneal epithelium in 1 out 3 eyes examined. According to these findings, the test item was classified as negative for GHS Category 1 for non-extreme pH (2 < pH < 11.5) detergents and surfactants.

Therefore, in accordance with the Regulation (EC) No. 1272/2008, the test item RICOIL EN is classified in the category “no prediction can be made”. It 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.

As a conservative approach and in the absence of additional testing (in vitro and/or in vivo), the substance is classified as irritant to eye (category 2).

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 irritation/corrosion

The combination of an in vitro skin irritation test according to OECD 439 and an in vitro skin corrosion test according to OECD 431 results in the classification of Reaction products of “Fatty Acids, C16-18 and C18-unsatd., Me esters; sulfuric acid and sodium hydroxide” in Category 2 “Irritating to skin” requiring the hazard statement “H315: Causes skin irritation” with the signal word “Warning”.

eye irritation

The results obtained under n in vitro eye irritation test according to OECD 438 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).

However, these results are in contradiction with the results obtained with skin irritation in vitro (OECD 439) and skin corrosion in vitro (OECD 431). Indeed, the product has been classified in category 2 "Irritant" for its effects on the skin.

A histopathological examination was performed. Following the application of the test item RICOIL EN, there was very slight (1/2) erosion of the superficial part of the superficial corneal epithelium in 1 out 3 eyes examined. According to these findings, the test item was classified as negative for GHS Category 1 for non-extreme pH (2 < pH < 11.5) detergents and surfactants.

Therefore, in accordance with the Regulation (EC) No. 1272/2008, the test item RICOIL EN is classified in the category “no prediction can be made”. It 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.

As a conservative approach and in the absence of additional testing (in vitro and/or in vivo), the substance is classified in Category 2 “Eye irritation” requiring the hazard statement “H319: Causes serious eye irritation” with the signal word “Warning”.