Amidation products of C16-18 (even numbered), C18 unsaturated fatty acids esters with 1,1'-iminodipropan-2-ol

Endpoint summary

Description of key information

Skin Corrosion - In Vitro

Not corrosive in the Reconstructed Human Epidermis (RHE) Test

Skin Irritation - In Vitro

Non irritant in the human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)).

Skin Irritation/Corrosion - In Vivo

Not irritating to the skins of rabbits (4 hour semi-occulsive application)

Eye Irritation/corrosion - In Vivo

Not irritating in New Zealand White Rabbits.

Eye Irritation - In Vitro

No induction of ocular irritation in the BCOP test.

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:

04 April 2016 to 08 April 2016

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:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method (adopted 28 July 2015).

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch: RC-1045Study specific test item informationPurity/composition correction factor: No correction factor requiredChemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)CAS Number: 1454803-04-3Test item handling: No specific handling conditions required

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

other: Not applicable

Details on animal used as source of test system:

Test system EpiDerm Skin Model (EPI-200, Lot no.: 23697 kit U and T). The model consists of normal, 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts. Source MatTek Corporation, Ashland MA, U.S.A.

Justification for test system used:

Rationale Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TissuesOn the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium. DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM medium, serum-free supplied by MatTek Corporation. MTT medium MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.Environmental conditions All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 69 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 36.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Control samples:

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

50 μl

Duration of treatment / exposure:

Two tissues were used for a 3-minute exposure to MLA-3202 and two for a 1-hour exposure.

Duration of post-treatment incubation (if applicable):

Tissues were incubated for 3 hours at 37°C in air containing 5% CO2.

Number of replicates:

2 per application time/group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour treatment

Value:

86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute treatment

Value:

67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MLA-3202 was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that MLA-3202 did not interfere with the MTT endpoint. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with MLA-3202 compared to the negative control tissues was 67% and 86% respectively. Because the mean relative tissue viability for MLA-3202 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment MLA-3202 is considered to be not corrosive. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 7%. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 5%, indicating that the test system functioned properly.

Mean absorption in thein vitroskin corrosion test with MLA-3202

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	2.144	2.239	2.192	±	0.067	2.033	2.081	2.057	±	0.034
MLA-3202	1.439	1.496	1.467	±	0.040	1.764	1.756	1.760	±	0.006
Positive control	0.146	0.152	0.149	±	0.004	0.141	0.218	0.179	±	0.055

SD = Standard deviation

Duplicate exposure are indicated by A and B.

In this table the values are corrected for background absorption (0.0405). Isopropanol was used to measure the background absorption.

 

Mean tissue viability in thein vitroskin corrosion test with MLA-3202

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
MLA-3202	67	86
Positive control	7	9

 

Coefficient of Variation between tissue replicates

	3 minute	1 hour
Negative control	4.3	2.3
MLA-3202	3.8	0.5
Positive control	3.9	35.5

CV (%) = 100 – [(lowest OD570/highest OD570) x 100%]

 

INDIVIDUAL OD MEASUREMENTS AT 570 NM

	3-minute application (OD570)		1-hour application (OD570)
	A	B	A	B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3	2.1973 2.1816 2.1752	2.3017 2.2773 2.2608	2.1016 2.0743 2.0458	2.1050 2.1339 2.1257
MLA-3202 OD570measurement 1 OD570measurement 2 OD570measurement 3	1.4956 1.4680 1.4756	1.5285 1.5558 1.5246	1.8073 1.7824 1.8248	1.8057 1.7708 1.8121
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3	0.1862 0.1874 0.1870	0.1939 0.1910 0.1934	0.1827 0.1816 0.1792	0.2583 0.2639 0.2537

OD – Optical density

Duplicate exposure are indicated by A and B.

 

HISTORICAL CONTROL DATA FOR IN VITRO SKIN CORROSION STUDIES

	Negative control		Positive control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (% viability)	1-hour treatment (% viability)
Range	1.324 – 2.615	1.361 – 2.352	0.172 – 0.56	0.057 – 0.277	6 – 22	3 – 12
Mean	1.86	1.86	0.18	0.13	10.67	7.17
SD	0.24	0.22	0.10	0.05	3.9	2.36
n	65	67	64	61	30	30

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of December 2012 to December 2015.

Interpretation of results:

GHS criteria not met

Conclusions:

Finally, it is concluded that this test is valid and that MLA-3202 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

This objective of the study was to evaluate the corrosivity potential of MLA-3202 using a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of MLA-3202 was tested through topical application for 3 minutes and 1 hour. This method was designed to be compatible with the following:

-OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method” (adopted 28 July 2015)

-Method B.40 of Commission Regulation (EC) No. 440/2008

 

Batch RC-1045 of MLA-3202 was a clear amber-red liquid. MLA-3202 was applied undiluted (50 μl) directly on top of the skin tissue.

 

The positive control had a mean relative tissue viability of 7% after 3 minutes of exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of 20 - 100% viability, the Coefficient of Variation between tissue replicates was ≤ 5%, indicating that the test system functioned properly.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with MLA-3202 compared to the negative control tissues was 67% and 86%, respectively. Because the mean relative tissue viability for MLA-3202 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment MLA-3202 is considered to be not corrosive.

 

Finally, it is concluded that this test is valid and that MLA-3202 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2016 to 17 May 2016

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:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (adopted 28 July 2015).

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test ". Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch: RC-1045Study specific test item informationPurity/composition correction factor: No correction factor requiredChemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)CAS Number: 1454803-04-3Test item handling: No specific handling conditions required

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: three-dimensional human epidermis model

Source strain:

other: Not applicable

Details on animal used as source of test system:

Test systemEPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-019).This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.SourceSkinEthic Laboratories, Lyon, France.

Justification for test system used:

RationaleIn the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

Tissues On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 24 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.MTT medium MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml). Environmental conditions All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 67 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 – 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Control samples:

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The liquid test item was applied undiluted (25 μl) directly on top of the tissue.

Duration of treatment / exposure:

Exposure period of 15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

Skin tissues were incubated for 42 hours at 37°C.

Number of replicates:

3 per test group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes treatment

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with MLA-3202 compared to the negative control tissues was 94%. Since the mean relative tissue viability for MLA-3202 was above 50% MLA-3202 is considered to be non-irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 12%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 15%, indicating that the test system functioned properly.

Mean absorption in thein vitroskin irritation test with MLA-3202

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.755	0.924	0.968	0.882	±	0.112
MLA-3202	0.974	0.745	0.762	0.827	±	0.128
Positive control	0.115	0.136	0.069	0.107	±	0.035

OD = optical density

SD = Standard deviation

Triplicate exposure are indicated by A, B and C.

In this table the values are corrected for background absorption (0.041). Isopropanol was used to measure the background absorption.

 

Mean tissue viability in thein vitroskin irritation test with MLA-3202

	Mean tissue viability (percentage of control)
Negative control	100
MLA-3202	94
Positive control	12

 

Individual OD measurements at 570 nm

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	0.8261 0.7665	0.9820 0.9482	1.0665 0.9509
MLA-3202 OD570measurement 1 OD570measurement 2	1.0080 1.0226	0.7824 0.7907	0.8794 0.7268
Positive control OD570measurement 1 OD570measurement 2	0.1570 0.1562	0.1895 0.1653	0.1112 0.1086

OD = Optical density

Triplicate exposure are indicated by A, B and C.

 

Historical control data forin vitroskin irritation studies

	Negative control (absorption; OD570)	Positive control (absorption; OD570)	Positive control (viability; %)
Range	0.676 – 1.336	0.028 – 0.408	2.46 – 42.99
Mean	1.06	0.16	14.78
SD	0.14	0.11	9.29
n	115	115	115

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of December 2012 to December 2015.

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that the test is valid and that MLA-3202 is non-irritant in the in vitro skin irritation test under the experimental conditions described in the report.

Executive summary:

The objective of this study was to evaluate the potential of MLA-3202 to induce skin irritation using a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of MLA-3202 was tested through topical application for 15 minutes.

 

The study procedures described in this report were designed to be compatible with the most recent OECD and EC guidelines.

 

Batch RC-1045 of MLA-3202 was a clear amber-red liquid. MLA-3202 was applied undiluted (25 μl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 94%. Since the mean relative tissue viability for MLA-3202 was above 50% after 15 ± 0.5 minutes treatment MLA-3202 is considered to be non-irritant.

 

The positive control had a mean cell viability of 12% after 15 ± 0.5 minutes exposure. The absolute mean OD₅₇₀ (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 15%, indicating that the test system functioned properly.

 

Finally, it is concluded that this test is valid and that MLA-3202 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 August 2016 to 08 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Weight of Evidence Analysis -In Vivo Study In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed, prior to the start of this in vivo eye irritation study in the rabbit. As recommended in the test guidelines, all available information was evaluated (e.g. existing human and animal data, literature, item data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity) and in vitro, ex vivo and in vivo tests) to determine the need for in vivo eye testing.The in vitro Bovine Corneal Opacity and Permeability (BCOP) study (project 514868) showed that the test item was non-irritating. This negative BCOP result should be confirmed in an in vivo eye irritation study. Based on the available information, it was concluded that there was the need to perform this in vivo eye irritation study in rabbit in order to establish the possible eye irritating properties of the test item.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No.405, "Acute Eye Irritation / Corrosion", Paris, 2012.

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B5: "Acute Toxicity: Eye Irritation/Corrosion". Official Journal of the European Union No. L142, May 2008, including most recent amendments.

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.2400, Acute Eye Irritation. Office of Prevention, Pesticides and Toxic Items (7101), EPA 712-C-98-195, August 1998.

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

other: JMAFF No. 8147

Version / remarks:

Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000; including the most recent partial revisions.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Specific details on test material used for the study:

Study Specific Test Item InformationQuality system of the CoA: GLPPurity/composition correction factor: No correction factor requiredTest item handling: No specific handling conditions requiredStability at higher temperatures: StableChemical name (IUPAC), synonym or trade name: Amides, tallow, N,N-bis(2-hydroxypropyl)CAS Number: 1454803-04-3pH: 6-7Specific gravity/density: 0.9394

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species: Albino rabbit, New Zealand White, (SPF-Quality). Recognized by international guidelines as the recommended test system (e.g. EC, OECD) Source: Charles River France, L’Arbresle, France Number of animals: 3 Males Age and body weight: At start of dosing, the animals were between 12 and 24 weeks old and body weights were at least 1.5 kg. Identification: Earmark. Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the eyes were free from any abnormality. Animal Husbandry Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study. Accommodation: Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm). Acclimatization period was at least 5 days before start of treatment under laboratory conditions. Diet: Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) approximately 100 grams per day. Hay (TecniLab-BMI BV, Someren, The Netherlands) and wooden sticks (Swedish aspen wood, Bioservices, Uden, The Netherlands) were available during the study period. Water: Free access to tap water. Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

Each animal was treated by instillation of 0.1 mL of the test item

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 1 week later, after considering the degree of eye irritation observed in the first animal.

Details on study design:

Pre-emptive Pain Management One hour prior to instillation of the test item, buprenorphine (Buprenodale®, Dechra Ltd., Stoke-on-Trent, United Kingdom) 0.01 mg/kg was administered by subcutaneous injection in order to provide a therapeutic level of systemic analgesia. Five minutes prior to instillation of the test item, two drops of the topical anesthetic alcaine 0.5% (SA Alcon-Couvreur NV, Puurs, Belgium) were applied to both eyes. Treatment Each animal was treated by instillation of 0.1 mL of the test item, in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test item. The other eye remained untreated and served as the reference control. Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area. After the final observation, the animals were sacrificed by intra-venous injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).Observations Mortality/Viability: Twice daily. Toxicity: At least once daily. Body Weight: Day of treatment (prior to instillation) and after the final observation. Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test item. The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to a numerical scoring system (see “Any other information” for details).Necropsy: No necropsy was performed according to study plan.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

mean

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

Irritation Instillation of approximately 0.1 mL of MLA-3202 into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and/or discharge. The irritation had completely resolved within 24 hours in two animals and within 48 hours in the other animal. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test item instillation revealed no corneal epithelial damage. Corrosion There was no evidence of ocular corrosion.

Other effects:

Coloration / Remnants No staining of (peri) ocular tissues by the test item was observed and no test item remnants were seen. Toxicity / Mortality No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual Eye Irritation Scores

Animal	Time after dosing	Cornea			Iris	Conjunctivae			Comments
		Opacity (0-4)	Area (0-4)	Fluor area (%)^2	(0-2)	Redness (0-3)	Chemosis (0-4)	Discharge (0-3)
164^1	1 hour 24 hours 48 hours 72 hours	0 0 0 0	0 0 0 0	0	0 0 0 0	1 0 0 0	0 0 0 0	0 0 0 0	- - - -
175	1 hour 24 hours 48 hours 72 hours	0 0 0 0	0 0 0 0	0	0 0 0 0	1 0 0 0	0 0 0 0	1 0 0 0	- - - -
178	1 hour 24 hours 48 hours 72 hours	0 0 0 0	0 0 0 0	0	0 0 0 0	1 1 0 0	1 0 0 0	1 0 0 0	- - - -

¹Sentinel,²Green staining after fluorescein treatment (percentage of total corneal area)

 

Mean Value Eye Irritation Scores

Animal	Mean 24, 48 and 72 hours
	Corneal opacity	Iris	Conjunctivae
			Redness	Chemosis
164 175 178	0.0 0.0 0.0	0.0 0.0 0.0	0.0 0.0 0.3	0.0 0.0 0.0

 

Animal Specifications

Animal	Sex	Age at start (weeks)	Body weights (grams)
			Prior to application	At termination
164 175 178	Female Female Female	13 13 13	3052 2421 2671	3088 2670 2769

 

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results, MLA-3202 does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).

Executive summary:

Acute eye irritation/corrosion study with MLA-3202 in the rabbit.

 

The study was carried out based on the guidelines described in:

OECD No.405 (2012) "Acute Eye Irritation / Corrosion"

EC, No 440/2008, B5: "Acute Toxicity: Eye Irritation/Corrosion"

EPA, OPPTS 870.2400 (1998): "Acute Eye Irritation"

JMAFF Guidelines (2000), including the most recent revisions.

 

Single samples of 0.1 mL of MLA-3202 were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours after instillation.

 

Instillation of MLA-3202 into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and/or discharge. The irritation had completely resolved within 24 hours in two animals and within 48 hours in the other animal.

 

Based on these results, MLA-3202 does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments). 

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 July 2016

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:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 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”(adopted July 26, 2013).

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Specific details on test material used for the study:

Test item 207258/AIdentification MLA-3202Appearance Clear amber-red liquidBatch RC-1045Purity/Composition UVCBTest item storage At room temperatureStable under storage conditions until 17 February 2019 (expiry date)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter. Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test. Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

750 μl

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

Duration of post- treatment incubation (in vitro):

The corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.

Number of animals or in vitro replicates:

3 replicates/treatment

Details on study design:

Study design Preparation of corneas The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C. Cornea selection and Opacity reading After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.Treatment of corneas and opacity measurements The medium from the anterior compartment was removed and 750 μl of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.Opacity measurement The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. See “Any other information” for the calculation.The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.Application of sodium fluorescein Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C. Permeability determinations After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μl of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

cornea opacity score

Value:

>= 1.3 - <= 2.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Value:

> 1.3 - < 3.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from -1.3 to -0.4. The individual positive control in vitro irritancy scores ranged from 53 to 73 for Ethanol. The corneas treated with the positive control item were turbid after the 10 minutes of treatment. The corneas treated with MLA-3202 showed opacity values ranging from 1.3 to 2.7 and permeability values ranging from -0.006 to 0.027. The corneas were translucent after the 10 minutes of treatment with MLA-3202. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 1.2 to 3.1 after 10 minutes of treatment with MLA-3202. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 62 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Summary of opacity, permeability andin vitroscores

Treatment	Mean Opacity^1	Mean Permeability^1	MeanIn vitroIrritation Score^1,2
Negative control	-1.0	0.010	-0.8
Positive control (Ethanol)	22.3	2.636	61.8
MLA-3202	2.2	0.006	2.3

¹Calculated using the negative control mean opacity and mean permeability values for the positive control and test item

²In vitroirritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value)

 

Opacity score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1	Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	4.0	2.6	-1.4		-1.0
	2.7	2.1	-0.6
	1.9^5	7.3^5	5.5^5
Positive control	3.0	21.3	18.4	18.4	22.3
	1.6	23.8	22.2	22.2
	2.2	28.4	26.2	26.2
MLA-3202	3.5	6.2	2.7	2.7	2.2
	3.0	4.3	1.3	1.3
	1.7	4.3	2.6	2.6

¹Final Opacity = Opacity after treatment – Opacity before treatment.

²Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control.

³In case the mean final opacity if the negative control is below zero, no correction will be made.

⁴Calculations are made without rounding off.

⁵Eye excluded from analysis (study plan deviation 1)

 

Permeability score individual values (uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control	1	0.004	0.005	0.005	0.005	0.005	0.010
	1	0.020	0.014	0.010	0.015	0.015
	1	0.001^2	-0.001^2	0.001^2	0.000^2	0.000^2
Positive control	6	0.398	0.400	0.399	0.399	2.394
	6	0.572	0.575	0.569	0.572	3.432
	6	0.377	0.374	0.377	0.376	2.256
MLA-3202	1	0.037	0.037	0.037	0.037	0.037
	1	0.001	0.005	0.004	0.003	0.003
	1	0.007	0.007	0.010	0.008	0.008

¹Calculations are made without rounding off

²Eye excluded from analysis (study plan deviation 1)

 

Permeability score individual values (corrected)

Treatment	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OD490Average	Negative control corrected final OD490	Average OD
Positive control	6	0.388	0.390	0.389	0.389	2.336	2.636
	6	0.562	0.565	0.559	0.562	3.374
	6	0.367	0.364	0.367	0.366	2.198
MLA-3202	1	0.027	0.027	0.027	0.027	0.027	0.006
	1	-0.009	-0.005	-0.006	-0.006	-0.006
	1	-0.003	-0.003	0.000	-0.002	-0.002

1 OD₄₉₀values corrected for the mean final negative control permeability (0.010).

2 Calculations are made without rounding off.

 

In Vitroirritancy score

Treatment	Final Opacity^2	Final OD490^2	In VitroIrritancy Score^1
Negative control	-1.4	0.005	-1.3
	-0.6	0.015	-0.4
	5.5^3	0.000^3	5.5^3
Positive control	18.4	2.336	53.4
	22.2	3.374	72.8
	26.2	2.198	59.2
MLA-3202	2.7	0.027	3.1
	1.3	-0.006	1.2
	2.6	-0.002	2.6

¹In vitroirritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

²Positive control and test tem are corrected for the negative control

³Eye excluded from analysis (study plan deviation 1)

 

Historical control data for the BCOP studies

	Negative control			Positive control
	Opacity	Permeability	In vitroIrritancy Score	In vitroIrritancy Score
Range	-2.9 – 3.0	-0.016 – 0.029	-2.8 – 3.0	35.8 – 72.9
Mean	0.25	0.00	0.34	56.58
SD	1.07	0.01	1.18	12.51
n	51	43	45	21

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of February 2015 to May 2016.

Interpretation of results:

GHS criteria not met

Conclusions:

MLA-3202 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.3 after 10 minutes of treatment.Since MLA-3202 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

Evaluation of the eye hazard potential of MLA-3202 using the Bovine Corneal Opacity and Permeability test (BCOP test).

 

The report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of MLA-3202 was tested through topical application for 10 minutes.

 

The study procedures described in this report were based on the most recent OECD guideline.

 

Batch RC-1045 of MLA-3202 was a clear amber-red liquid. The test item was applied as it is (750 μl) directly on top of the corneas.

 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 62 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

MLA-3202 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.3 after 10 minutes of treatment.

 

Since MLA-3202 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin Corrosion - In Vitro

This study was designed to be compatible with the following:

-OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method” (adopted 28 July 2015)

-Method B.40 of Commission Regulation (EC) No. 440/2008

 

Batch RC-1045 of MLA-3202 was a clear amber-red liquid. MLA-3202 was applied undiluted (50 μl) directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 7% after 3 minutes of exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of 20 - 100% viability, the Coefficient of Variation between tissue replicates was ≤ 5%, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with MLA-3202 compared to the negative control tissues was 67% and 86%, respectively. Because the mean relative tissue viability for MLA-3202 was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment MLA-3202 is considered to be not corrosive.

 

Finally, it is concluded that this test is valid and that MLA-3202 is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Skin Irritation - In Vitro

The objective of this study was to evaluate the potential of MLA-3202 to induce skin irritation using a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of MLA-3202 was tested through topical application for 15 minutes.

The study procedures described in this report were designed to be compatible with the most recent OECD and EC guidelines.

 

Batch RC-1045 of MLA-3202 was a clear amber-red liquid. MLA-3202 was applied undiluted (25 μl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 94%. Since the mean relative tissue viability for MLA-3202 was above 50% after 15 ± 0.5 minutes treatment MLA-3202 is considered to be non-irritant.

 

The positive control had a mean cell viability of 12% after 15 ± 0.5 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 15%, indicating that the test system functioned properly.

 

Finally, it is concluded that this test is valid and that MLA-3202 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Skin Irritation/Corrosion - In Vivo

Primary skin irritation/corrosion study with MLA-3202 in the rabbit (4-hour semi-occlusive application).

The study was carried out based on the guidelines described in:

OECD No.404, "Acute Dermal Irritation/Corrosion" (2015)

EC, No 440/2008; B4: "Acute Toxicity: Dermal Irritation/Corrosion".

US EPA, OPPTS 870.2500 (1998), Acute Dermal Irritation.

JMAFF Guidelines (2000), including the most recent revisions.

 

Three rabbits were exposed to 0.5 mL of MLA-3202 by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours and 7 and 14 days after exposure for the first animal and 1, 24, 48 and 72 hours after exposure for the other two animals.

 

Exposure to MLA-3202 resulted in very slight to well-defined erythema and very slight oedema in the treated skin areas of the three rabbits. One animal showed scaliness after 7 days. The skin irritation resolved within 14 days after exposure for the first animal and 48 hours after exposure for the other two animals.

 

Based on the results MLA-3202 does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Eye Irritation - In Vivo

The study was carried out based on the guidelines described in:

OECD No.405 (2012) "Acute Eye Irritation / Corrosion"; EC, No 440/2008, B5: "Acute Toxicity: Eye Irritation/Corrosion"; EPA, OPPTS 870.2400 (1998): "Acute Eye Irritation"; JMAFF Guidelines (2000), including the most recent revisions.

 

Single samples of 0.1 mL of MLA-3202 were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours after instillation.

 

Instillation of MLA-3202 into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and/or discharge. The irritation had completely resolved within 24 hours in two animals and within 48 hours in the other animal.

 

Based on these results, MLA-3202 does not have to be classified and has no obligatory labelling requirement for eye irritation. 

Acute Eye Irritation - In Vitro

Evaluation of the eye hazard potential of MLA-3202 using the Bovine Corneal Opacity and Permeability test (BCOP test).

 

The eye damage of MLA-3202 was tested through topical application for 10 minutes.

 

Batch RC-1045 of MLA-3202 was a clear amber-red liquid. The test item was applied as it is (750 μl) directly on top of the corneas.

 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 62 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

MLA-3202 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.3 after 10 minutes of treatment.

 

Since MLA-3202 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Justification for classification or non-classification

Skin Corrosion

Based on the results, MLA-3202 does not have to be classified and has no obligatory labelling requirement for skin corrosivity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments). 

Skin Irritation

Based on the results MLA-3202 does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Eye irritation/Eye Damage

Based on the results, MLA-3202 does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).