LUMIPOD

Endpoint summary

Description of key information

Conclusion OECD 439:

The mean viability of the tissues treated with the test item is 1%. According to the prediction model described in the 4" revised edition of the GHS, the test item is irritant.

 

Conclusion OECD 431: 

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item L-Leucine, reaction products with 1,4:3,6 dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride 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.

 

Conclusion OECD 437:

According to the defined grading scale, no prediction can be made for the classification of the test item L-Leucine- reaction products with 1-4:3-6-dianhydro-D-glucitol- iso-Pr alc. and octanoyl chloride - Batch : 200122016662 concerning eye irritation or serious eye damage.

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:

08/11/2011 - 22/11/2011

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

other: PK2KTU06

Vehicle:

not specified

Details on test system:

Test system:
The present study has been performed using SkinEthic Reconstituted Human Epidermal model (RHE) from Skin Ethic laboratories (Lyon, France), which is validated for skin irritation testing.
The statement regarding validity of in vitro tests for skin irritation testing from the ECVAM Scientific Advisory Committee (ESAC) is joined in annex 1.
Epidermal tissues were received on November 15, 2011, by Elodie Bombard, at day 18 of the differentiation process (air-lifted cultures).
Epidermal tissue viability and responsiveness were ensured by the supplier, as well as expiry dates.
The technical and quality data sheet from SkinEthic Laboratories presented in annex 2 confirmed the good shape and viability of the epidermis. Before the beginning of the experiments, the Study Director checked all documents and the integrity of tissues and culture media (maintenance medium and growth medium).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

16 + 0.5 µl

Duration of treatment / exposure:

42min (+-1min) at room temperature.

Duration of post-treatment incubation (if applicable):

RHE were then incubated for 42h (+-1h) at 37°C, 5% CO, for recovery.

Number of replicates:

3 replicates by test item and reference

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three runs with two replicates each

Value:

ca. 1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results

1. MTT interference

These data were presented in the specific study reports.

 

2. Acceptance criteria and test validity

Negative control (NC) acceptance criteria: The NC data meet the acceptance criteria if the mean OD value of the 3 tissues is >= 1.2 at 570 nm. The standard deviation value is considered as valid if it is =< 18%.

Positive control (PC) acceptance criteria: The PC data meet the acceptance criteria if the mean viability, expressed as %of the NC, is < 40 % and the standard deviation value is =< 18%.

Batch acceptance criteria: All test items data from one batch are considered as valid if both negative and positive controls data fulfill the above criteria requirements.

Acceptance criteria	Standard deviation value	Validity of the test
NC acceptance criteria	o.7%	Yes
PC acceptance criteria	0.2 %	Yes
Batch acceptance criteria	/	Yes

3. MU viability testing

A MTT assay was performed to measure the viability of RHE after a 42 min exposure to the test and reference items and a 42h recovery period.

The mean OD of the three tissues exposed to the negative control (PBS) was set to represent icio % of tissue viability and the results were expressed as a percentage of the negative control viability.

According to the 4^th revised edition of the Globally Harmonized System of Classification and Labelling of Chemicals (61-15; United Nation (New York and Geneva, 2011)), the prediction model is as follows:

Criteria for in vitro interpretation	Classification GHS United Nation
Mean tissues viability is =< 50 %	Category 2, irritant (I)
Mean tissues viability is > 50 %	No Category, non irritant (NI)

 

Conclusions:

The mean viability of the tissues treated with the test item is 1%. According to the prediction model described in the 4" revised edition of the GHS, the test item is irritant.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19/03/2020 - 29/04/2020

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: 3-D, fully differentiated human epidermis equivalent, reconstructed from normal human primary epidermal keratinocytes

Cell source:

other: not specified

Source strain:

other: not specified

Vehicle:

not specified

Details on test system:

The 0.6 cm2 reconstituted epidermis (epiCS, Cell Systems - Batch No.100-AJ0806-1) were received on 28 April 2020. The four additional killed control tissues (epiCS, Cell Systems - Batch No.100 AJ0456-1 frozen on 27 March 2020) were defrozen the day of the treatment, on 29 April 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-AJ0846). The culture plates were incubated at 37+2°C, 5% CO2 during 19 hours and 45 minutes before treatment. Just before the treatment, the culture medium was replaced by a new culture medium (Cell Systems, Batch No. 305-AJ0846).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50µl

Duration of treatment / exposure:

3 minutes at room temperature and during 1 hour at 37°C+-1°C, 5% +-1% CO2

Duration of post-treatment incubation (if applicable):

The skin sample was placed for 3 hours at 37°C 1°C, 5% CO2 into an MTT solution of 300 uL concentration, except the 4 tissues for the non-specific control which were placed in MTT assay medium (Cell Systems Batch No. 303- AJ0846).
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)

Number of replicates:

3 replicates by test item and reference

Irritation / corrosion parameter:

% tissue viability

Value:

>= 78.69 - <= 81.14

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

RESULTS
3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 78.69% and 81.14%, versus 58.06% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N).

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item L-Leucine, reaction products with 1,4:3,6 dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride 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 L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride after topical administration on in vitro human reconstituted epidermis (epics®, supplied by CellSystems). 

 

Method

The test item L-Leucine, reaction products with 1,4:3,6-dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride was applied as supplied, at the dose of 50 uL, 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. Additionally, 4 killed Human skin model surfaces were treated under the same experimental conditions in order to generate non-specific MTT reduction. The experimental protocol was established in accordance with the 0.E.C.D. Test Guideline No. 431 dated 18 June 2019.

 

Results

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 78.69% and 81.14%, versus 58.06% and 0.5%, respectively, with the positive control item (potassium hydroxide 8N). 

 

Conclusion

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item L-Leucine, reaction products with 1,4:3,6 dianhydro-D-glucitol, iso-Pr alc. and octanoyl chloride 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 (corrosive)

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:

16/03/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificat n°2019/19

Species:

other: bovine cornea

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

Corneas of calves aged less than 8 months. Calves eyes were collected in the slaughterhouse of Sobeval Boulazac 24759 - France, in a short time after slaughtering of the animals, and transported in a Hanks's buffered saline solution with antibiotic to the laboratory At reception, the eyes were carefully examined under lighting and these showing a visible (scratches, pigmentation, neo-vascularization) defect were eliminated.
For each selected eye, an incision with a scalpel was practiced at the level of the scleral ring by means of scissors.
Approximately 2 to 3 mm of scleral ring were left to facilitate the further handlings.
Corneas were immersed in Hanks's medium at room temperature.
Then the corneas were used at receipt for the test.
The demonstration of the skill of the laboratory is performed using the test substances recommended by the guideline followed.

Vehicle:

not specified

Controls:

yes

Amount / concentration applied:

750 +- 75 mg of the test substance

Duration of treatment / exposure:

The treatment lasted (time of exposure) was 10 minutes.
After the end of the exposure period, precisely signalled by the ringing of the chronometer, the test item and controls are removed from the anterior compartment and the epithelium washed at least three times (or until no visual evidence of test item can be observed) with EMEM containing phenol red.

Duration of post- treatment incubation (in vitro):

The opacity of each cornea was measured at 2 times, just before treatment with the test item (measurement called OPTO) and 2 hours after the end of the 10 minutes contact (measurement called OPT2).

Number of animals or in vitro replicates:

Not applicable.
Triplicate corneas for each timepoint and tested substance (test item, vehicle control, positive control).

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of three runs with two replicates each

Value:

>= 15.1 - <= 16.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

According to the defined grading scale, no prediction can be made for the classification of the test item L-Leucine- reaction products with 1-4:3-6-dianhydro-D-glucitol- iso-Pr alc. and octanoyl chloride - Batch : 200122016662 concerning eye irritation or serious eye damage.

Executive summary:

PRINCIPLE OF THE STUDY
The aim of the study was to assess quantitatively the irritant potential of a test item after application to the isolated calf cornea.
The assessment was based on the measurement of two parameters: the opacity and permeability of the cornea whose deteriorations reflected the damage of the tissue.
The test item was let in contact with the isolated cornea for 10 minutes.
On the basis of the results obtained, an In Vitro Irritancy score (IVIS) was calculated to classify the irritancy level of the test item.

EXPERIMENTAL STARTING DATE AND EXPERIMENTAL COMPLETION DATE: March 16, 2020

RESULTS:

Test item	Tested concentration	IVIS 10 minutes Mean ± standard deviation
L-Leucine- reaction products with 1- 4:3-6-dianhydro-D-glucitol- iso-Pr alc. and octanoyl chloride Batch : 200122016662	as supplied	15.8 ± 0.7

No prediction can be made for the classification of the test item L-Leucine- reaction products with 1-4:3-6-dianhydro-D-glucitol- iso-Pr alc. and octanoyl chloride - Batch ; 200122016662 concerning eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)