Registration Dossier

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Administrative data

Description of key information

Skin Irritation
The skin irritancy of the test substance, TM 11-213 was determined according to OECD Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model. TM 11-213, was classified was classified as an irritant.
Eye irritation
The eye irritancy potential of the test substance TM 11-213 was assessed as irritating according to OECD Test Guideline 405 using an in vivo method.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Testing was conducted between 03 September and 09 September 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
Refer to principles of method if other than guideline
Principles of method if other than guideline:
Deviations from Study Plan:

Deviation No.1 (03 October 2013) Section 4.1 Direct MTT reduction.
An assessment found the test item was able to directly reduce MTT. Therefore an additional
procedure using water-killed tissues was performed during the determination of skin irritation
potential. However the results obtained showed a negligible degree of interference due to direct
reduction of MTT occurred. It was therefore considered unnecessary to use the results of the
water-killed tissues for quantitative correction of results or for reporting purposes.

Deviation No.2 (03 October 2013)
The control group’s for this study were shared with study 41302525.
The control groups raw data will be filed with study 41302525.
This deviation was considered not to affect the purpose or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Species:
other: EPIKSIN in vitro Reconstructed Human Epidermis (RHE) Model
Strain:
other: Not applicable
Details on test animals or test system and environmental conditions:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 03 September 2013
EpiSkinTM Tissues (0.38cm2) lot number : 13-EKIN-030
Maintenance Medium lot number : 13-MAIN3-037
Assay Medium lot number : 13-ESSC-031
Type of coverage:
other: Not applicable
Preparation of test site:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: Not applicable
Duration of treatment / exposure:
15-Minute exposure period and 42 hours post-exposure incubation period.
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT:
MTT Salt Metabolism, Cell Viability Assay:

The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.

One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of thecellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure:

10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours.
Untreated MTT solution was used as a control.

If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues.

This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test substance like viable tissues. Water-killed tissues were prepared by placing untreated EPISKINTM tissues in a 12-well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37 °C, 5% CO2 in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30 °C) for up to 6 months. Before use each tissue was thawed by placing in 2.0 mL of maintenance medium for approximately 1 hour at room temperature.

In addition to the normal test procedure, each MTT reducing test substance was applied to a water-killed tissue. In addition, one water-killed tissue remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue.


PRE-INCUBATION (DAY 0: TISSUE ARRIVAL):
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarosegel and the insert:

Tissues Satisfactory : Yes
Temperature Indicator Color Satisfactory : Yes
Agar Medium Color Satisfactory : Yes

2.0 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of three wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

MAIN TEST:
APPLICATION OF TEST ITEM AND RINSING (DAY 2):
2.0 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SDS solution was re-spread with a
pipette tip to maintain the distribution of the SDS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. Residual test item remained on the tissues after rinsing. The rinsed tissues were transferred to the second column of 3 wells containing 2.0 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.


MTT LOADING/FORMAZAN EXTRACTION (DAY 3):
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.

2.0 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry.

A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at
562 nm (without a reference filter) using the Anthos 2001 microplate reader.
Irritation / corrosion parameter:
other: other: relative mean viability
Value:
12.5
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 minutes. Remarks: Irritating to Skin . (migrated information)

Direct MTT Reduction

An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin irritation potential. However, the results obtained showed no degree of interference due to direct

reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

 

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 12.5 % after a 15-Minute exposure period and 42 hours post-exposure incubation period. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal.

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.0% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.0 %. The positive control acceptance criterion was therefore satisfied.

The mean OD562for the negative control treated tissues was 0.863 and the standard deviation value of the percentage viability was 9.8 %. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 4.0 %. The test item acceptance criterion was therefore satisfied.

Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

OD562 of

tissues

Mean OD562

of triplicate

tissues

± SD of

OD562

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

± SD of

Relative

mean

viability (%)

Negative

Control Item

0.797

0.863

0.084

92.4

100*

9.8

0.958

111.0

0.834

96.6

Positive Control Item

0.069

0.069

0.009

8.0

8.0

1.0

0.061

7.1

0.078

9.0

Test Item

0.146

0.108

0.034

16.9

12.5

4.0

0.099

11.5

0.079

9.2

SD = Standard deviation

*The mean viability of the negative control tissues is set at 100 %

Interpretation of results:
Category 2 (irritant)
Remarks:
Migrated information Cause skin irritation Criteria used for interpretation of results: EU
Conclusions:
The test item, TM 11-213, was classified as irritant. The following classification criteria apply:
EU CLP and UN GHS Hazard statement H315 “Causes Skin Irritation” Category 2.
EU DSD (67/548/EEC) Irritant requires symbol “Xi” risk phrase R38 “Irritating to Skin”.
Executive summary:

The skin irritancy of the test substance, TM 11-213 was determined according to OECD Guideline 439 using the EPISKIN™ Reconstructed Human Epidermis Model. The relative mean viability of the test item treated tissues was 12.5 % after a 15-Minute exposure period and 42 hours post-exposure incubation period. This results shows that the substance is a skin irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 02 March 2015 and 06 April 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 405 using an in vivo method and in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Three New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.93 to 3.22 kg and were 12 to 20 weeks old. After an acclimatization period of at least 5 days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light (06:00 to 18:00) and 12 hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.


Justification
The rabbit is the preferred species of choice as historically used for irritation studies and is specified in the appropriate test guidelines.
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye remained untreated and was used for control purposes
Amount / concentration applied:
0.1 mL of the test item
Duration of treatment / exposure:
The test item was not removed
Observation period (in vivo):
21 days
Number of animals or in vitro replicates:
Three
Details on study design:
Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

Initially, a single rabbit was treated. A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre dose anesthesia of ocular anesthetic (two drops of 0.5% tetracaine hydrochloride) was applied to each eye.

A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made.

Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.

After consideration of the ocular responses produced in the first treated animal, two additional animals were similarly treated.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation.

Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Any clinical signs of toxicity, if present, were also recorded.

Additional observations were made on Days 7, 14 and 21 to assess the reversibility of the ocular effects.

Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Irritation parameter:
iris score
Remarks:
Male 74958
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0.33
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
iris score
Remarks:
Male 75009
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0.33
Max. score:
2
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
iris score
Remarks:
Male 75010
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
0.66
Max. score:
2
Reversibility:
fully reversible within: 72 hours
Irritation parameter:
conjunctivae score
Remarks:
Male 74958
Basis:
mean
Remarks:
Redness
Time point:
other: 24, 48 and 72 hours
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 21 days
Irritation parameter:
conjunctivae score
Remarks:
Male 75009
Basis:
mean
Remarks:
Redness
Time point:
other: 24, 48 and 72 hours
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
Male 75010
Basis:
mean
Remarks:
Redness
Time point:
other: 24, 48 and 72 hours
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Remarks:
Male 74958
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 21 days
Irritation parameter:
chemosis score
Remarks:
Male 75009
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
chemosis score
Remarks:
Male 75010
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritant / corrosive response data:
Red colored staining of the fur around the treated eye was noted in one animal 1 hour after treatment.

No corneal effects were noted during the study.

Iridial inflammation was noted in all treated eyes 1 and 24 hours after treatment and in one treated eye at the 48 Hour observation.

Moderate conjunctival irritation was noted in all treated eyes 1 hour after treatment and at the 24, 48 and 72 Hour observations. Minimal conjunctival irritation was noted in one treated eye at the 7 and 14 Day observations.

Two treated eyes appeared normal at the 7 Day observation and one treated eye appeared normal at the 21 Day observation.
Other effects:
Body Weight
All animals showed expected gain in body weight during the study.

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex

74958Male

75009Male

75010Male

IPR= 0

IPR = 0

IPR = 0

Time After Treatment

1
Hr

24
Hr

48
Hr

72
Hr

7
Dy

14
Dy

21
Dy

1
Hr

24
Hr

48
Hr

72
Hr

7
Dy

1
Hr

24
Hr

48
Hr

72
Hr

7
Dy

CORNEA

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

E = Degree of Opacity

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

F = Area of Cornea Involved

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Score (E x F) x 5

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

IRIS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

D

1

1

0

0

0

0

0

1

1

0

0

0

1

1

1

0

0

Score (D x 5)

5

5

0

0

0

0

0

5

5

0

0

0

5

5

5

0

0

CONJUNCTIVAE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A = Redness

2

2

2

2

1

1

0

2

2

2

2

0

2

2

2

2

0

B = Chemosis

2

2

2

2

1

1

0

2

2

2

2

0

2

2

2

2

0

C = Discharge

2Sf

2

1

1

0

0

0

2

2

2

1

0

2

2

2

1

0

Score (A + B + C) x 2

12

12

10

10

4

4

0

12

12

12

10

0

12

12

12

10

0

Total Score

17

17

10

10

4

4

0

17

17

12

10

0

17

17

17

10

0

IPR= Initial pain reaction                   

Hr = Hour            

Dy = Days

Sf = Red colored staining of the fur around the treated eye

Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number
and Sex

Individual Total Scores At:

1
Hour

24
Hours

48
Hours

72
Hours

7
Days

14
Days

21
Days

74958Male

17

17

10

10

4

4

0

75009Male

17

17

12

10

0

-

-

75010Male

17

17

17

10

0

-

-

Group Total

51

51

39

30

4

4

0

Group Mean Score

17.0

17.0

13.0

10.0

1.3

1.3

0.0

- =  Observation not required - considered to be zero for calculation of Group Mean Score

Individual Body Weights and Body Weight Change

Rabbit Number
and Sex

Individual Body Weight (kg)

Body Weight Change (kg)

74958Male

Day 0

Day 21

0.15

2.99

3.14

75009Male

Day 0

Day 7

0.08

3.22

3.30

75010Male

Day 0

Day 7

0.17

2.93

3.10

 

Interpretation of results:
Category II
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item produced a maximum group mean score of 17.0 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item was classified as Category 2A (irritating to eyes) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

The test item was also classified as Irritating to eyes (Category 2) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. It is reasonable to assume that the Signal Word “Warning” and the Hazard Statement “H319: Causes serious eye irritation” are therefore required.
Executive summary:

The eye irritation potential of the test substance, TM 11-213, was assessed according to OECD Test Guideline 405 using an in vivo method as classified - Category 2 (irritating to eyes).

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin Corrosion/Irritation

Skin corrosion is defined as the production of irreversible damage to the skin, namely visible necrosis through the epidermis and into the dermis following the application of a test substance for up to 4 hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs and, by the end of observations at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia and scars. Skin irritation is defined as the production of reversible damage to the skin following the application of a test substance for up to 4 hours. Several factors are considered in determining the corrosion and irritation potential of substances before in vivo testing is undertaken.

The potential for chemical induced skin corrosion is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. To this end, an in vitro study was performed to assess the potential for skin corrosion using the EPISKINTMin vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.

Validation studies have shown that tests employing human skin models are able to reliably distinguish between known skin corrosives and non-corrosives (Bothamet al.,1995, Barrettet al., 1998 and Fentemet al., 1998). At its 10th Meeting, held on 31 March 1998 at ECVAM, Ispra, Italy, the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed the EPISKINTMmodel as scientifically validated for use as a replacement for the animal test. The EPISKINTMmodel is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items.

The EPISKINTMmodel is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

The procedure followed is based on the recommended EpiSkinTMSkin Corrosivity Test protocol INVITTOX No118. The test item is applied topically to the stratum corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKINTMmodel. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control.

 

This study was designed to be compatible with the procedures indicated by OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

 

The MTT solution containing the test item turned blue. This was taken to indicate that the test item reduced MTT and the MTT viability assay was therefore performed in parallel on viable and water-killed tissues.

The relative mean viabilities of the test item TM-11-213 at 3 minutes exposure was 135.7%, at 60 minutes exposure was 104.4% and at 240 minutes exposure was 114.1%.  

 

The relative mean tissue viability for the positive control treated tissues was 3.3% relative to the negative control treated tissues following the 240-Minute exposure period, with a mean OD562for the negative control treated tissues of 0.818. The results for the negative and positive control acceptance criterion was satisfied and the test item classified as non-corrosive to the skin.

 

The test item was assessed for skin irritation using the EPISKINTMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours (Fentemet al., 2001, Zuanget al., 2002, Cotovioet al., 2005, Portes et al., 2002 and Hartung, 2007). As described above, the principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1αin the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.

 

Following a full validation study, the EpiSkinTMreconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Test items were applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

This study was designed to be compatible with the procedures indicated by the OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010).

 

An assessment found the test item was able to directly reduce MTT. There was no interference due to direct reduction of MTT. The relative mean viability of the test item was 12.5% after a 15-Minute exposure period and 42 hours post-exposure incubation period. The test item TM-11-213 was classified as an irritant.

Eye Irritation

Serious eye damage is defined as the production of tissue damage in the eye, or serious physical decay of vision following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application. Eye irritation means the production of changes in the eye following application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application. The classification system for substances involves a tired testing and evaluation scheme and a number of factors are considered in determining eye irritation or serious eye damage.

 

Initially, a study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. Two endpoints, namely decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

This study was designed to be compatible with the procedures indicated by the OECD Guidelines for the Testing of Chemicals No. 437 (2009) “Bovine Corneal Opacity and Permeability Assay”.

The results showed that the corneas treated with the test item were clear post incubation and therefore the test item TM-11-213 was considered not to be an ocular corrosive or severe irritant.

 

Further to this, the eye irritation potential of the test item TM-10-213 was assessed using the SkinEthic reconstructed Human Corneal Epithelium model after a treatment period of 10 minutes.

 

The SkinEthic HCE model consists of transformed human corneal epithelial cells that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, histologically, the mucosa of the human eye (Nguyen et al., 2003). The test item is applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum free and chemically defined medium.

 

The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic HCE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Cytotoxicity is determined by the reduction of MTT to formazan by viable cells in the test item treated tissues (quantitative measurement of tissue viability) relative to the negative control.

 

Relative mean tissue viability of ≤ 60 % results in a prediction of ocular irritancy. After a 10 minute exposure, the relative mean viability of the test item treated tissues was 69.5% and TM 11-213 is therefore considered to be non-irritant.

 

An in vivo study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit and was designed to be compatible with OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 02 October 2012).

Results from the study showed that no corneal effects were noted during the study. Iridial inflammation was noted in all treated eyes 1 and 24 hours after treatment and in one treated eye at the 48‑Hour observation. Moderate conjunctival irritation was noted in all treated eyes 1 hour after treatment and at the 24, 48 and 72‑Hour observations. Minimal conjunctival irritation was noted in one treated eye at the 7 and 14‑Day observations. Two treated eyes appeared normal at the 7‑Day observation and one treated eye appeared normal at the 21‑Day observation.

 

The test item produced a maximum group mean score of 17.0 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system. The test item was classified as Category 2A (irritating to eyes) according to the Globally Harmonized System of Classification and Labelling of Chemicals. The test item was also classified as Irritating to eyes (Category 2) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted on the target substance using an appropriate in vitro method according to internationally recognised test guidelines.

Justification for selection of eye irritation endpoint:
The study was conducted on the target substance in vivo, in an appropriate test species and according to internationally recognised test guidelines.

Effects on skin irritation/corrosion: irritating

Effect level: empty Endpoint conclusion: Adverse effect observed

Justification for classification or non-classification

Skin Corrosion/Irritation

Using a quantitative MTT assessment, the classification of corrosivity potential is based on relative viabilities for each exposure time. At 3 minutes treatment time a relative mean tissue viability (percentage of negative control) of <35 indicates corrosivity. Likewise at 3/60 minutes and 60/240 minutes treatment time, a relative mean tissue viability (percentage of negative control) of ≥35/<35 also indicates corrosivity. A relative mean tissue viability of ≥35 at 240 minutes treatment however is an indication that the substance is non-corrosive. The relative mean viability of the test substance, TM 11-213 at 3, 60 and 240 minutes was 135.7, 104.4 and 114.1 % respectively and therefore is classified as non-corrosive to the skin.

Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period and a 42-hour post-exposure incubation period. The test item is considered an irritant if the relative mean tissue viability is ≤ 50 % and a non-irritant if the relative mean tissue viability is > 50 %. The relative mean viability of the tissues treated with the test item TM 11-213 was 12.5 % and therefore is classified as an irritant.

Eye Irritation

The classification system for substances involves a tiered testing evaluation scheme and a number of factors are considered in determining eye irritation or serious eye damage. From predictions a test item is an irritant if the relative mean tissue viability ≤ 60 % and a non-irritant if the relative mean tissue viability ≥ 60 %. The relative mean viability of the tissues treated with the test item TM 11-213 was 69.5 % after a 10 minute exposure period and is therefore considered to be non-irritant.

 

A study was performed to assess the ocular irritancy potential of the test item, TM 11-213 to isolated bovine cornea, using an in vitro irritancy score. If the test item induces an in vitro irritancy score ≥ 55.1, it is defined as an ocular corrosive or severe irritant and will be labelled EU CLP/UN GHS Category 1. The in vitro irritancy score of the test item, TM 11-213 was 5.2 and was therefore considered not to be an ocular corrosive or severe ocular irritant.

 

When in vivo studies are conducted, classification as an eye irritant is dependent on the severity and duration of the effects.  If, when the substance is applied to the eye of an animal the substance produces corneal opacity ≥1, and/or conjunctival redness ≥2 and/or conjunctival oedema (chemosis) ≥2 in at least two of the three animals, the test item is considered as irritating to eyes. This is calculated as the mean scores following grading at 24, 48 and 72 hours after installation of the test material.

In vivo testing, of test item, TM 11-213, demonstrated effects over the initial 72 hours of testing. Where they occurred, these effects were fully resolved within 21 days. No effects were noted for corneal opacity. Conjunctival redness and conjunctival oedema were above the threshold values for classification. The test substance was therefore classified as irritating to the eye. The test item was classified as Category 2A (irritating to eyes) according to the Globally Harmonized System of Classification and Labelling of Chemicals, and Irritating to eyes (Category 2) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.