Glycerides, tall-oil mono-

Endpoint summary

Description of key information

Skin Corrosion - In vitro

LUMULSE GMT-K is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Skin Irritation - in vitro

LUMULSE GMT-K is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Eye irritation - in vitro

Under the conditions of the study, the test item LUMULSE GMT-K showed no effects on the cornea of the bovine eye. The calculated IVIS is 1.35.

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:

19. Feb. 2018 to 22. Feb. 2018

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:

OECD Guidelines for the Testing of Chemicals Part 431, adopted 29. Jul. 2016 “In vitro Skin Corrosion: reconstructed human epidermis (RHE) test method”

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Commission Regulation (EU) No.440/2008, EU-Method B.40 BIS. “IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST“ dated 30. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

Specification
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.

Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SCT
Day of delivery: 20. Feb. 2018
Batch: 25882

Justification for test system used:

The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion and are cytotoxic to the underlying cell layers.

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-Tests
Nylon mesh compatibility
First, the test item was tested for possible reaction with the nylon mesh which is used to ensure sufficient contact with the tissue surface. 50 μL of the liquid test item were brought onto a nylon mesh on a microscope slide. No reaction with the mesh was visible after 1 hour incubation at room temperature.

Assessment of Coloured or Staining Test Items
It was tested whether the test item develops a colour without MTT addition. 50 μL of the test item were given in a test tube with 0.3 mL demineralised water and incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour.
The resulting solution was colourless, therefore no binding capacity had to be tested.

Assessment of Direct Reduction of MTT by the Test Item
The test item was also tested for the ability of direct MTT reduction. To test for this ability, 50 μL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour. Untreated MTT solution was used as control.
The MTT solution did not change its colour within 1 hour. Therefore, direct MTT reduction had not taken place and no data correction was necessary.

Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the assay medium directly before use.
The tissue plate was brought out of the fridge 1 hour before the treatment.
The assay medium was warmed in the water bath to 37 ± 1°C.

Description of the Method
Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour (pre-incubation).
For each experiment (“3 minutes” and “1 hour”), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 μL assay medium, the other 12 with 300 μL MTT solution. One additional plate was left empty. The plates were stored in the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2.
For each experiment (“3 minutes” and “1 hour”), two 6-well-plates for the assay were used.
After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 μL demineralised water, two wells as positive controls with 50 μL potassium hydroxide solution and two other wells for testing the test item.
The liquid test item was applied without preparation (50 μL).
At the start of each experiment (application of negative controls), a stop watch was started.
After the respective incubation time (“3 minutes” and “1 hour”) at 37 ± 1°C and 5.0 ±0.5% CO2, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were immediately moved to the wells containing MTT medium, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT solution for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
After this time, the MTT solution was aspirated and replaced by DPBS. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 hours at room temperature.
Afterwards, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, three replicates with 200 μL solution (each) were pipetted into a 96-wellplate which was read in a plate spectrophotometer at 570 nm.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The liquid test item was applied without preparation (50 μL).

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

The tissues were incubated with MTT solution for 3 hours

Number of replicates:

For each experiment, two 6-well-plates for the assay were used.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

99.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

87.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Assessment and Validity
Corrosivity of the Test Item
The mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 99.6% and 87.8% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item LUMULSE GMT-K is considered to be not corrosive.

Validity
The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 1.8 (3 minutes) resp. 1.9 (1 hour).
The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The mean value of relative tissue viability was 6.6%
Values for negative control and for positive control were within the range of historical data of the test facility.
Therefore the experiment is considered valid.

Measured Values

As blank, the optical density of isopropanol was measured in 12 wells of the 96-well-plate.

The measured values and their mean are given in the following table:

Absorbance values blank isopropanol (OD 570 nm)

Replicate	1	2	3	4	5	6	Mean 0.038
Absorbance	0.040	0.038	0.040	0.040	0.037	0.038
Replicate	7	8	9	10	11	12
Absorbance	0.039	0.038	0.037	0.038	0.038	0.037

 

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values (OD 570 nm)

Incubation	Negative Control		Test item		Positive Control
	Tissue 1	Tissue 2	Tissue 1	Tissue 2	Tissue 1	Tissue 2
3 min	1.800	1.787	1.758	1.802	0.486	0.475
	1.780	1.800	1.756	1.814	0.492	0.473
	1.785	1.769	1.760	1.790	0.487	0.473
1 h	1.884	1.961	1.635	1.698	0.161	0.156
	1.860	1.916	1.631	1.743	0.160	0.165
	1.864	1.927	1.641	1.700	0.161	0.166

 

From the measured absorbances, the mean absorbance of isopropanol was subtracted. The corrected mean and relative standard deviation (RSD) of the two tissues were also calculated.

Mean Absorbance Values of the 3 Minutes Experiment

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.750	1.720	0.450
Mean – blank (tissue 2)	1.747	1.764	0.435
Mean of the two tissues	1.749	1.742	0.443
RSD	0.1%	1.8%	2.3%

 

Mean Absorbance Values of the 1 h Experiment

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.831	1.597	0.122
Mean – blank (tissue 2)	1.896	1.675	0.124
Mean of the two tissues	1.864	1.636	0.123
RSD	2.5%	3.4%	1.0%

 

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of mean tissue viability were calculated in comparison to the mean of the negative controls:

% Tissue Viability

Test Item	Positive Control	Incubation
99.6%	25.3%	3 min
87.8%	6.6%	1 h

 

Interpretation of results:

GHS criteria not met

Conclusions:

LUMULSE GMT-K is considered non-corrosive to skin.
The mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 99.6% and 87.8% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item LUMULSE GMT-K is considered to be not corrosive.
The values of the negative control met the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals, thus showing the quality of the tissues.
The positive control has met the validity criterion too, thus ensuring the validity of the test system.
For these reasons, the result of the test is considered valid.

Executive summary:

Title of Study: Determination of Skin Corrosion Potential of LUMULSE GMT-K in the Reconstructed Human Epidermis (RHE) Test Method following OECD Guideline 431 and EU Method B.40-BIS

 

Findings and Results:

One valid experiment was performed.

Two tissues of the human skin model EpiDermTM were treated with the test item LUMULSE GMT-K for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan.

Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were within the requiredacceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.8 (3 minutes experiment) and 1.9 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 6.6% for the 1 hour treatment.

The mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 99.6% and 87.8% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item LUMULSE GMT-K is considered to be not corrosive.

 

Therefore, LUMULSE GMT-K is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19. Feb. 2018 to 23. Feb. 2018

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:

OECD Guideline for the Testing of Chemicals, Version 439, adopted 28. July 2015, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending Regulation (EC) No. 761/2009, Annex III, EU method B.46 “IN VITRO SKIN IRRITATION: RECONSTRUCTED HUMAN EPIDERMIS MODEL TEST”, adopted 06. Jul. 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

Specification
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

Origin
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-200-SIT
Day of delivery: 20. Feb. 2018
Batch no.: 25882

Justification for test system used:

In accordance with test guidelines.

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre - Tests
Nylon mesh compatibility
The test item was tested for possible reaction with the nylon mesh, which is used to ensure sufficient contact with the tissue surface. 30 μL of the test item were pipetted onto a nylon mesh on a microscope slide.
No reaction with the nylon mesh was visible after an exposure time of 1 hour.

Assessment of Coloured or Staining Test Items
It was tested whether the test item develops a colour without MTT addition. 30 μL test item were given in a test tube with 0.3 mL H2O demin. and incubated at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour.
The resulting solution was colourless, therefore no binding capacity had to be tested.

Assessment of Direct Reduction of MTT by the Test Item
The test item was tested for the ability of direct MTT reduction. To test for this ability, 30 μL test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at 37 ± 1°C and 5.0 ± 0.5% CO2 for 1 hour. Untreated MTT medium was used as control.
The MTT solution did not change its colour within 1 hour. Therefore, direct MTT reduction by the test item had not taken place and no data correction was necessary.

Pre-Incubation of Tissues
All working steps were performed under sterile conditions. For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ± 1°C and 5 ± 0.5% CO2 for 1 hour.
After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ± 1°C and 5.0 ± 0.5% CO2 for 18 hours and 55 minutes.

Treatment
One plate (3 tissues) was used as negative control; each tissue was treated with 30 μL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used as positive control; each tissue was treated with 30 μL 5% SDSsolution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used for treatment with the test item; 30 μL test item were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 23 hours and 25 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.

Medium Renewal
After post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate.
Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 18 hours and 50 minutes for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2.

MTT Assay
After a total incubation time of 42 hours and 15 minutes, a 24-well-plate was prepared with 300 μL freshly prepared MTT-solution in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours at 37 ± 1°C and 5.0 ± 0.5% CO2.
After this time, the MTT-solution was aspirated and replaced by DPBS buffer. This was then aspirated, too, and replaced several times.
At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate.
Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken (120 rpm) for 2 hours at room temperature.
After 2 hours, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 μL solution (each) were pipetted into a 96-wellplate which was read in a plate spectrophotometer at 570 nm.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 μL test item were applied

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

23 hours and 25 minutes

Number of replicates:

3 replicates per test system (tets item, negative and positive controls)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item

Value:

93.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Skin Irritation Potential of the Test Item
The mean value of relative tissue viability of the test item was reduced to 93.5% after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-irritant to skin.

Validity and Acceptability
All validity criteria were met.
Values for negative control and for positive control were within the range of historical data of the test facility.

Measured Values

As blank, the optical density of isopropanol was measured in 8 wells of the 96-well-plate.

The measured values and their mean are given in the following table:

Absorbance values blank isopropanol (OD 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.041	0.039	0.041	0.041	0.039	0.040	0.041	0.040	0.040

 

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values negative control, test item and positive control (OD 570 nm)

Designation	Measurement	Negative Control	LUMULSE GMT-K	Positive Control
Tissue 1	1	1.670	1.568	0.089
	2	1.727	1.604	0.102
Tissue 2	1	1.674	1.482	0.091
	2	1.667	1.492	0.104
Tissue 3	1	1.629	1.601	0.088
	2	1.618	1.605	0.091

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol. The mean of the three tissues was also calculated.

Mean Absorbance Values

Designation	Negative Control	LUMULSE GMT-K	Positive Control
Mean – blank (tissue 1)	1.659	1.546	0.056
Mean – blank (tissue 2)	1.631	1.447	0.058
Mean – blank (tissue 3)	1.584	1.563	0.050
Mean of the three tissues	1.625	1.519	0.055

 

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control:

% Tissue Viability

Designation	LUMULSE GMT-K	Positive Control
% Tissue viability (tissue 1)	95.1%	3.4%
% Tissue viability (tissue 2)	89.0%	3.6%
% Tissue viability (tissue 3)	96.2%	3.1%
% Tissue viability (mean)	93.5%	3.4%
± SD of mean tissue viability (%)	3.9%	0.3%

 

Validity and Acceptability

Validity criteria and results are stated in the following table:

Validity

Criterion	Demanded	Found
OD of negative control	≥ 0.8 and ≤ 2.8	1.6
% tissue viability of positive control SDS	≤ 20% of negative control	3.4%
SD of mean viability of the tissue replicates (%)	≤ 18%	2.3% (negative control) 0.3% (positive control) 3.9% (test item)

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item LUMULSE GMT-K is considered as non-irritant to skin.
After the treatment, the mean value of relative tissue viability was reduced to 93.5%. This value is above the threshold for skin irritation (50%).
The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8.
The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system.
Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%).
For these reasons, the result of the test is considered valid.

Executive summary:

Title of Study: Determination of Skin Irritation Potential of LUMULSE GMT-K in the Reconstructed human Epidermis (RhE) Test Method following EU-Method B.46 resp. OECD 439

 

Findings and Results:

One valid experiment was performed.

Three tissues of the human skin model EpiDermTM were treated with LUMULSE GMT-K for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance values was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.6. The positive controlshowed clear irritating effects. The mean value of relative tissue viability was reduced to3.4% (required:≤20%).

The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤18%).

After the treatment with the test item, the mean value of relative tissue viability was reduced to 93.5%. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

 

Therefore, LUMULSE GMT-K is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

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:

23. Jan. 2018

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)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Method No. 437, adopted 09. Oct. 2017: “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

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Commission Regulation (EU) 2017/735 amending Regulation (EC) No. 440/2008, EU Method B.47 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, adopted 14. Feb. 2017

Deviations:

no

Principles of method if other than guideline:

OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160: “GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE IRRITANTS”; 25. Oct. 2011

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

Species: Bos primigenius Taurus (fresh bovine corneas)
Origin: Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container within 1 hour.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 μL negative control solution, test item and positive control were applied to each replicate.

Duration of treatment / exposure:

Exposure time of the test item on the corneas was 10 minutes at 32 ± 1 °C.

Duration of post- treatment incubation (in vitro):

2 hours at 32 ± 1 °C (post-incubation).

Number of animals or in vitro replicates:

For each treatment group (negative control solution, test item and positive control), three replicates were used.

Details on study design:

Preparations
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

Experimental Parameters
Date of treatment: 23. Jan. 2018
Incubation time: 10 min.
Negative control: HBSS
Positive control: Dimethylformamide (undiluted)

Method Description
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, test item and positive control were applied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:

Closed Chamber Method
The “closed chamber method” is used for liquid substances.
The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 μL of the appropriate liquid through the refill hole in the holder on the cornea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item.
Exposure time of the test item on the corneas was 10 minutes at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation).
After post-incubation time, the cMEM without phenol red was renewed in both chambers.
Then, the final opacity value of each cornea was recorded. The cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 4 mg/mL) was added to the front chamber.

Permeability Test
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas.
For the closed chamber method, a sodium fluorescein solution with a concentration of 4 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean IVIS

Value:

1.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

In the negative control, no signs of eye irritation were observed.
The positive control induced serious eye damage, which would be classified as GHS category I.
The test item LUMULSE GMT-K showed no effects on the cornea of the bovine eye. The calculated IVIS is 1.35.
The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Opacity and Permeability Values

The illuminance (unit: LUX) values which were measured before and after exposure are given in the following table:

Illuminance Values

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
(I) Measured values before exposure	983	990	990	1008	998	957	956	986	977
(I) Measured values after exposure	957	968	962	975	912	913	268	286	362

Rep. = Replicate

 

The values in the following tables present the calculated opacity values, according to evaluation:

Opacity Values negative Control

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.82	3.51	3.51
Opacity after exposure	4.99	4.49	4.76
Opacity Difference	1.17	0.98	1.25
Mean Opacity Difference	1.13

Rep. = Replicate

 

Opacity Values Test Item and Positive Control

Parameter	Test item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	2.74	3.60	4.99	5.04	3.68	4.08
Opacity after exposure	4.17	7.18	7.13	119.16	109.18	77.98
Opacity Difference	1.43	3.58	2.14	114.12	105.50	73.90
Opacity Difference corrected	0.29	2.45	1.01	112.99	104.36	72.77
Mean Opacity Difference corrected	1.25			96.71

Rep. = Replicate

 

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:

Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.036
2. Measurement	0.038
3. Measurement	0.036
Mean	0.037

 

Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1. Measurement	0.061	0.050	0.036	0.037	0.043	0.086	1.367	0.630	0.852
2. Measurement	0.061	0.048	0.031	0.037	0.040	0.084	1.411	0.647	0.881
3. Measurement	0.057	0.051	0.036	0.038	0.040	0.085	1.390	0.648	0.871
1. Measurement – blank	0.0243	0.0133	- 0.0007	0.0003	0.0063	0.0493	1.3303	0.5933	0.8153
2. Measurement – blank	0.0243	0.0113	- 0.0057	0.0003	0.0033	0.0473	1.3743	0.6103	0.8443
3. Measurement – blank	0.0203	0.0143	- 0.0007	0.0013	0.0033	0.0483	1.3533	0.6113	0.8343
Mean of each replicate	0.0230	0.0130	- 0.0023	0.0007	0.0043	0.0483	1.3527	0.6050	0.8313
Mean of the 3 replicates	0.0112			--			--
Corrected	--	--	--	-0.0106	-0.0069	0.0371	1.3414	0.5938	0.8201
Corrected mean of the 3 replicates	--			0.0066			0.9484

Rep. = Replicates

 

IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

IVIS

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	1.52	1.30	14.60%
	1.17
	1.21
Test Item LUMULSE GMT-K	0.14	1.35	83.16%
	2.35
	1.56
Positive Control DMF undiluted	133.11	110.48	21.85%
	113.27
	85.07

Note: The high relative standard deviation of the IVIS of test item is due to mathematical reasons, as the respective means are very small.

Interpretation of results:

GHS criteria not met

Conclusions:

This in vitro study was performed to assess corneal damage potential of LUMULSE GMTK by quantitative measurements of changes in opacity and permeability in a bovine cornea.
The test item LUMULSE GMT-K was applied onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.
The test item was tested neat.
Under the conditions of this test, the test item LUMULSE GMT-K showed no effects on the cornea of the bovine eye. The calculated IVIS is 1.35.
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.
The negative control (HBSS) and the positive control (undiluted dimethylformamide) have met the validity criteria.
No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Executive summary:

Title of Study: Evaluation of LUMULSE GMT-K in the Bovine Corneal Opacity and Permeability (BCOP) Test Method following OECD Guideline 437 resp. EU Method B.47

 

Findings and Results:

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test item LUMULSE GMT-K was applied onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS is 1.30.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 110.48.

Under the conditions of this study, the test item LUMULSE GMT-K showed no effects on the cornea of the bovine eye. The calculated IVIS is 1.35.

According to OECD Guideline No. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion - in vitro

One valid experiment was performed.

Two tissues of the human skin model EpiDermTM were treated with the test item LUMULSE GMT-K for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control, 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan.

Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were within the requiredacceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals thus showing the quality of the tissues. The OD was 1.8 (3 minutes experiment) and 1.9 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 6.6% for the 1 hour treatment.

The mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 99.6% and 87.8% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item LUMULSE GMT-K is considered to be not corrosive.

Therefore, LUMULSE GMT-K is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test Method.

Skin Irritation - in vitro

One valid experiment was performed.

Three tissues of the human skin model EpiDermTM were treated with LUMULSE GMT-K for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance values was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.6. The positive controlshowed clear irritating effects. The mean value of relative tissue viability was reduced to3.4% (required:≤20%).

The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤18%).

After the treatment with the test item, the mean value of relative tissue viability was reduced to 93.5%. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

Therefore, LUMULSE GMT-K is considered non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Eye Irritation - in vitro

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test item LUMULSE GMT-K was applied onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS is 1.30.

Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 110.48.

Under the conditions of this study, the test item LUMULSE GMT-K showed no effects on the cornea of the bovine eye. The calculated IVIS is 1.35.

Justification for classification or non-classification

As the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment, the test item LUMULSE GMT-K is considered to be not corrosive and classification is not required for skin corrosion.

After the treatment with the test item, the mean value of relative tissue viability was reduced to 93.5%. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin and classification for skin irritation is not required.

According to OECD Guideline No. 437 (Oct. 2017), a substance with an IVIS ≤ 3 requires no classification for eye irritation or serious eye damage.