Registration Dossier

Administrative data

Description of key information

The test substance, TM 10-202, was assessed for skin corrosion and irritation and for eye irritation according to internationally recognized guidelines both in in vitro and in vivo tests.  Results indicated that the test item was considered not to be corrosive or a skin irritant. The test substance did not meet the requirements of classification as an eye irritant. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 29 October 2013 and 12 November 2013.
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 404 using an in vivo method and in compliance with GLP.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
rabbit
Strain:
New Zealand White
Details on test animals 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 2.12 to 2.34 kg and were twelve to twenty weeks old.
After an acclimatization period of at least five 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 twelve hours continuous light (06:00 to 18:00) and twelve 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.
Type of coverage:
occlusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
0.5 mL of the test item was applied directly to the skin

Test Item Formulation and Experimental Preparation
For the purpose of the study the test item was used as supplied. The absorption of the test item was not determined
Duration of treatment / exposure:
Four hours
Observation period:
14 days
Number of animals:
Three
Details on study design:
On the day before the test each of a group of three rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
On the day of the test a suitable test site was selected on the back of each rabbit. A quantity of 0.5 mL of the test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.
Four hours after application the corset and patches were removed from each animal and any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.
Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored.
Any other skin reactions and clinical signs of toxicity, if present, were also recorded.
Additional observations were made on Days 7 and 14 to assess the reversibility of skin reactions.
Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.
Irritation parameter:
other: Erythema/Eschar Formation
Basis:
animal #1
Remarks:
73690 Male
Time point:
other: 24, 48 and 72 hours
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: loss of skin elasticity at 72 hour timepoint. At 7 days moderate desquamation noted.
Irritation parameter:
other: Erythema/Eschar Formation
Basis:
animal #2
Remarks:
73691 Male
Time point:
other: 24, 48 and 72 hours
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: loss of skin elasticity at 72 hour timepoint. At 7 days moderate desquamation and glossy skin noted.
Irritation parameter:
other: Erythema/Eschar Formation
Basis:
animal #3
Remarks:
73692 Male
Time point:
other: 24, 48 and 72 hours
Score:
2
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: loss of skin elasticity and light brown discoloration of the epidermis noted at 72 hours. At 7 days crust formation and adverse reaction prevented accurate evaluation of erythema.
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
73690 Male
Time point:
other: 24, 48 and 72 hours
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
73691 Male
Time point:
other: 24, 48 and 72 hours
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
73692 Male
Time point:
other: 24, 48 and 72 hours
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 14 days
Remarks on result:
other: At 7 days adverse reaction prevented accurate evaluation of edema
Irritant / corrosive response data:
Well-defined erythema and very slight edema were noted at all treated skin sites at the 24, 48 and 72-Hour observations. Very slight erythema was noted at two treated skin sites at the 7-Day observation.
Loss of skin elasticity was noted at all treated skin sites at the 72-Hour observation with light brown discoloration of the epidermis also noted at one treated skin site at the 72-Hour observation. Glossy skin and/or moderate desquamation was noted at two treated skin sites with crust formation, which prevented accurate evaluation of erythema and edema, noted at the remaining treated skin site at the 7-Day observation.
All treated skin sites appeared normal at the 14-Day observation
Other effects:
Body weight
All animals showed expected gain in body weight during the study.

Individual skin reactions

Skin reaction

Observation Time (following patch removal)

Individual scores – Rabbit No. & sex

Total

73690 Male

73691 Male

73692 Male

Erythema / Eschar Formation

Immediately

0

0

0

(0)

1 h

0

0

0

(0)

24 h

2

2

2

6

48 h

2

2

2

(6)

72 h

2Le

2Le

2LeBr

6

7 days

1D

1DG

?eCf

(2-6)

14 days

0

0

0

(0)

Edema Formation

Immediately

0

0

0

(0)

1 h

0

0

0

(0)

24 h

1

1

1

3

48 h

1

1

1

(3)

72 h

1

1

1

3

7 days

0

0

?2od

(0-4)

14 days

0

0

0

(0)

Sum of 24 and 72 h readings (S)

18

Primary irritation Index (S/6)

(18/6) = 3.0

Classification

Moderate irritant

( ) = Total values not used for calclation of primary dermal irritation index.

Le = Loss of skin elasticity

Br = Light brown discolouration of the epidermis

D = Moderate desquamation

G = Gloassy skin

Cf = Crust formation

?e = Adverse reaction prevented accurate evaluation of erythema

?od = Adverse reaction prevented accurate evaluation of edema

Individual body weights and body weight change

Rabbit No. & Sex

Individual Body Weight (kg)

Body Weight Change

(kg)

Day 0

Day 14

73690 Male

2.12

2.56

0.44

73691 Male

2.34

2.74

0.40

73692 Male

2.23

2.48

0.25

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item produced a primary irritation index of 3.0 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.
The test item meets the criteria for classification as a Mild irritant (Category 3) according to the Globally Harmonized System of Classification and Labelling of Chemicals.
The test item does not meet the criteria for classification according to the Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

The dermal irritation potential of the test substance, TM 10-202, was assessed as not irritating according to OECD Test Guideline 404 using an in vivo method.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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 11 November 2013 and 16 December 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 405 using an in vivo method and in compliance with GLP.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
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 2.44 to 2.83 kg and were twelve to twenty weeks old. After an acclimatization period of at least five 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 twelve hours continuous light (06:00 to 18:00) and twelve 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.
Vehicle:
unchanged (no vehicle)
Controls:
other: The left eye remained untreated for control purposes
Amount / concentration applied:
0.1 mL of the test item

Test Item Formulation and Experimental Preparation
For the purpose of the study the test item was used as supplied. The absorption of the test item was not determined.


Duration of treatment / exposure:
The test material was not removed.
Observation period (in vivo):
14 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 anaesthesia 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 according to the six point scale.
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 (Draize, J.H, 1977).
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 and 14 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:
cornea opacity score
Basis:
animal #1
Remarks:
73723 Male
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
73798 Male
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
73799 Male
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
4
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
73723 Male
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
2
Reversibility:
fully reversible within: 24 hours
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
73798 Male
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
73799 Male
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
2
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #1
Remarks:
73723 Male
Time point:
other: 24, 48 and 72 hours
Score:
2
Max. score:
3
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #2
Remarks:
73798 Male
Time point:
other: 24, 48 and 72 hours
Score:
1.67
Max. score:
3
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
Redness
Basis:
animal #3
Remarks:
73799 Male
Time point:
other: 24, 48 and 72 hours
Score:
0
Max. score:
3
Irritation parameter:
conjunctivae score
Remarks:
Chemosis
Basis:
animal #1
Remarks:
73723 Male
Time point:
other: 24, 48 and 72 hours
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 14 days
Irritation parameter:
conjunctivae score
Remarks:
Chemosis
Basis:
animal #2
Remarks:
73798 Male
Time point:
other: 24, 48 and 72 hours
Score:
1.67
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Remarks:
Chemosis
Basis:
animal #3
Remarks:
73799 Male
Time point:
other: 24, 48 and 72 hours
Score:
0.33
Max. score:
4
Reversibility:
fully reversible within: 48 hours
Irritant / corrosive response data:
No corneal effects were noted during the study.
Iridial inflammation was noted in one treated eye one hour after treatment.
Moderate conjunctival irritation was noted in all treated eyes one hour after treatment. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation noted in one treated eye at the 24 and 48-Hour observations. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in one other treated eye at the 72-Hour observation. Minimal conjunctival irritation was noted in one treated eye at the 7-Day observation.
One treated eye appeared normal at the 72-Hour observation, one other treated eye appeared normal at the 7-Day observation and the remaining treated eye appeared normal at the 14-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 No. & Sex

73723 Male

73798 Male

73799 Male

IPR = 0

IPR = 0

IPR = 0

Time after Treatment

1 h

24 h

48 h

72 h

7 days

14 days

1 h

24 h

48 h

72 h

7 days

1 h

24 h

48 h

72 h

Cornea

E = degree of opacity

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

Score = (E x F) x 5

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Iris

D

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Score (D x 5)

5

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctivae

A = redness

2

2

2

2

1

0

2

2

2

1

0

2

1

1

0

B = Chemosis

2

1

1

1

1

0

2

2

2

1

0

2

1

0

0

C = discharge

2

1

1

1

1

0

1

1

1

0

0

2

1

0

0

Score (A + B + C) x 2

12

8

8

8

6

0

10

10

10

4

0

12

6

2

0

Total Score

17

8

8

8

6

0

10

10

10

4

0

12

6

2

0

IPR = Initial pain reaction

Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit No. & Sex

Individual total scores at:

1 h

24 h

48 h

72 h

7 days

14 days

73723 Male

17

8

8

8

6

0

73798 Male

10

10

10

4

0

-

73799 Male

12

6

2

0

-

-

Group Total

39

24

20

12

6

0

Group Mean Score

13.0

8.0

6.7

4.0

3.0

0.0

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

Individual body weights and body weight changes

Rabbit No. & Sex

Individual Body Weight (kg)

Body Weight Change (kg)

73723 Male

Day 0

Day 14

0.31

2.83

3.14

73798 Male

Day 0

Day 7

0.08

2.59

2.67

73799 Male

Day 0

Day 3

0.07

2.44

2.51

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item produced a maximum group mean score of 13.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item, TM 10-202, does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

The test item, TM 10-202, does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

The eye irritation potential of the test substance, TM 10-202, was assesed as not an eye irritant according to OECD Test Guideline 405 using an in vivo method.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

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 EPISKINTM model 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 the following internationally accepted guidelines and recommendations:

·OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

 

Results showed that the test item did not reduce MTT and relative mean viabilities of the test item treated tissues were 104.5 % after 3 minutes exposure and 100.5 % after 240 minutes exposure. The test item should not therefore be classified as corrosive.

 

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, Porteset 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-1α in 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 following internationally accepted guidelines and recommendations:

·OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010).

 

The relative mean viability of the test item treated tissues was 85.0% after a 15-Minute exposure period and 42 hours post-exposure incubation period. From these results the test item was classified as non-irritant.

Further to this an in vivo study was performed to assess the irritancy potential of the test item following a single, 4-hour, semi-occluded application to the intact rabbit skin. This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

·OECD Guidelines for the Testing of Chemicals No. 404 “Acute Dermal Irritation/Corrosion” (adopted 24 April 2002)

A single 4-hour, semi-occluded application of the test item to the intact skin of three rabbits produced well-defined erythema and very slight edema at all treated skin sites at the 24, 48 and 72-Hour observations. Very slight erythema was noted at two treated skin sites at the 7-Day observation. A loss of skin elasticity was noted at all treated skin sites at the 72-Hour observation with light brown discoloration of the epidermis and also noted at one treated skin site at the 72-Hour observation. The appearance of glossy skin and/or moderate desquamation were noted at two treated skin sites with crust formation, which prevented accurate evaluation of erythema and edema, which was noted at the remaining treated skin site at the 7-Day observation. However, all treated skin sites appeared normal at the 14-Day observation, therefore according to OECD Test Guideline 402; the dermal irritation potential of test material was assessed as not irritating.

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 an in vitro study was performed to determine the eye irritation potential of the test item TM 10-202 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 100.5 % and TM 10-202 is therefore considered not classified for eye irritation.

 

Further to this, 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 the procedures indicated by the following internationally accepted guidelines and recommendations:

·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 but iridial inflammation was noted in one treated eye one hour after treatment. Moderate conjunctival irritation was noted in all treated eyes one hour after treatment. Moderate conjunctival irritation was noted in two treated eyes with minimal conjunctival irritation noted in one treated eye at the 24 and 48-Hour observations. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in one other treated eye at the 72-Hour observation. Minimal conjunctival irritation was noted in one treated eye at the 7-Day observation.

One treated eye appeared normal at the 72-Hour observation, one other treated eye appeared normal at the 7-Day observation and the remaining treated eye appeared normal at the 14-Day observation. Based on these results the eye irritation potential according to OECD Test Guideline 405 was assessed as not irritating.

 

No corneal effects were noted during the study, although there was some iridial inflammation and conjunctival irritation, which led to classification as a mild irritant (Class 4on a 1 to 8 scale) to the rabbit eye, according to a modified Kay and Calandra classification system. However, the test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals. The test item does not meet the criteria for classification 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 in vivo in an appropriate test species according to internationally recognised guidelines.

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

Justification for classification or non-classification

Skin Corrosion/Irritation

Two in vitro tests performed using EPISKINTM, indicated that the test item was considered not to be corrosive or a skin irritant. 

Further testing conducted in vivo demonstrated well-defined erythema and very slight edema at all treated skin sites at the 24, 48 and 72-Hour observations. Loss of skin elasticity, desquamations and discolouration were also observed, however all treated skin sites appeared normal and had reversed at the 14-Day observation.

One of the major criterion for a classification as an irritant is that at least two of three animals tested have a mean score of ≥ 2.3 - ≤ 4.0. The reversibility of skin lesions is also considered when evaluating irritant responses. When inflammation persists to the end of the observation period in two or more test animals, then a material may be considered as an irritant. As the in vivo test had a mean score of 0.0 the test item was classified as non-irritant to the skin.

 

Eye Irritation

The classification for substances involved a tired testing and evaluation scheme and a number of factors are considered in determining eye irritation or serious eye damage. pH extremes may produce serious eye damage and therefore the pH of the test substance is assessed before experiments are conducted. In this case a 90 % v/v aqueous preparation of the test item gave a pH of 7.0 immediately after preparation, and a pH of 7.0 after ten minutes, which is not within the parameters causing concern.

In in vitro tests, 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 10-202, was 100.5 % after a 10 minute exposure period and is therefore classified as non-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, the test item, TM10-202, demonstrated minor effects over the initial 72 hours of testing. Where they occurred, these effects were fully resolved within 14 days. In all cases the scores for corneal opacity, conjunctival redness and conjunctival oedema where less than the threshold values for classification. The test substance was therefore assessed as not irritating.