Registration Dossier

In the in vitro EPISKIN model test with SIKA Hardener MTJ the results indicated that the test item is neither corrosive nor irritating to the skin. In the in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eyes model with SIKA Hardener MTJ, ocular corrosion and severe irritation potential was not observed. In the in vivo Acute Eye Irritation Test the test item MTJ applied to the rabbits eye mucosa, caused slight to severe conjunctivae and slight cornea irrtant effects, fully reversible within 1 week.

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-05-29 to 2013-05-31

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:

22 July 210

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EPISKIN Standard Model

Strain:

other: EPISKIN Standard Model

Details on test animals or test system and environmental conditions:

not applicable

Type of coverage:

other: The test item was topically applied on the epidermal surface of two test skin units

Preparation of test site:

other: The epidermis units were preincubated overnight at 37 °C in an incubator.

Vehicle:

unchanged (no vehicle)

Controls:

other: Phosphate Buffered Saline (1 x PBS) and Sodium Dodecyl Sulphate (SDS, 5 % aq. solution) treated epidermis were used as negative and positive controls, respectively.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 20 µL of the unchanged test substance per epidermis unit

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes at room temperature.

Observation period:

After the incubation time, the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1 x solution to remove all of the test material from the epidermal surface. The EPISKIN-SM units were incubated for further 42 hours at 37 °C in an incubator with 5 % CO2.

Number of animals:

Three EPISKIN model units per test item and negative and positive control.

Details on study design:

TEST SITE
- Area of exposure: epidermal surface of skin unit (0.38 cm2 per unit)

REMOVAL OF TEST SUBSTANCE
- Washing: PBS 1 x solution
- Time after start of exposure: 15 min after treatment

SCORING SYSTEM: Cell viability

Irritation / corrosion parameter:

other: Cell Viability

Value:

109

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Basis: mean of three skin units. Time point: 15 min + 42 min post-exposure.

Interpretation of results:

not classified

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, SIKA Hardener MTJ is considered as non-irritant to skin and is therefore not classified.

Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item SIKA Hardener MTJ on reconstituted human epidermis in the EPISKIN model in vitro.

Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control.

The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50% when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item SIKA Hardener MTJ did not show significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, SIKA Hardener MTJ is considered as non-irritant to skin and is therefore not classified.

Endpoint conclusion:

no adverse effect observed (not irritating)

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-04-14-2014-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: S&K LAP Kft. 2173 Kartal, Császár út 135, HUNGARY
- Age at study initiation: Young adult rabbits
- Sex: Male
- Weight at study initiation: 2500 - 3118 g
- Housing: Animals were housed individually in metal cages.
- Diet: CRLT/ny, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: 11 days for first animal, 12 days for second animal and 13 days for third animal

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 10-15 changes/hour
- Photoperiod: Artificial light, from 6 a.m. to 6. p.m.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount applied: a weight of 0.1 mL of the undiluted test item was used for the study in pure state, in a single dose.

Duration of treatment / exposure:

single treatment

Observation period (in vivo):

1 hour after treatment
24 hours after treatment
48 hours after treatment
72 hours after treatment
1 week after treatment

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The eyes of the test animals were not washed out 24 hours after the application of test item, because the test substance was removed from the eye of the test animal by physiological mechanisms and the test item did not cause immediately severe irritation or corrosion after test item application.

SCORING SYSTEM:The eye irritation scores were evaluated according to the scoring system by Draize (1977) and OECD 405 (2nd October 2012).

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 1 week

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 1 week

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 1 week

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 1 week

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 72h

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48h

Irritation parameter:

other: discharge

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

other: discharge

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

other: discharge

Basis:

animal #3

Remarks:

Time point:

24/48/72 h

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 48 h

Irritant / corrosive response data:

One hour after treatment, some conjunctival hyperaemic blood vessels (score 1) were observed in all animals. The swelling was different from normal (score 1) in one animal and obvious swelling with partial eversion of lids (score 2) was found in two animals. 24 hours after treatment, some conjunctival hyperaemic blood vessels (score 1) were observed in all animals. The swelling was different from normal (score 1) in all animals. 48 hours after treatment, some conjunctival hyperaemic blood vessels (score 1) were observed in all animals. The swelling and the discharge was different from normal (score 1) in one animal. 1 week after the treatment the study was terminated, since no primary irritation symptoms occurred.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In conclusion, test item SIKA Hardener MTJ applied to the rabbits' eye mucosa, caused slight to severe conjunctivae and slight cornea irritant effects, fully reversible within 1 week.
According to Regulation (EC) No 1272/2008 (CLP), the test item has to be classified into “Category 2 (reversible effects on the eye/irritating to eyes)”on basis of cornea opacity.

Executive summary:

The acute eye irritation study of the test item SIKA Hardener MTJ was performed in three New Zealand White rabbits. The irritation effect of the test item was evaluated according to the Draize method (OECD No.: 405, 2012). The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A volume of 0.1 mL of the test item was used in pure state, as a single dose. The eyes of the test animals were not washed out after the application of test item. The eyes were examined at 1, 24, 48 and 72 hours, then 1 week after the application. One hour after the treatment, slight conjunctival redness, slight to moderate chemosis and moderate to severe discharge, as well as slight cornea opacity were observed. The area of cornea involved was greater than three quarters, up to the whole area in all animals. 72 hours after treatment slight redness and cornea opacity were recorded. The area of cornea involved was greater than three quarters, up to the whole area. 1 week after the treatment, all animals were free of symptoms, so the study was terminated. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

cornea opacity: 0.33, 1.00, 1.00

iris: 0.00, 0.00, 0.00

erythema: 1.00, 1.00, 1.00

chemosis: 0.33, 0.66, 0.33

discharge: 0.00, 1.33, 0.66

No systemic toxicity was observed on the day of the treatment and during the 1-week observation period. The body weight of animals corresponded to their species and age. Sign of pain and distress as discharge was observed in all animals. These symptoms were detected between treatment day and Day 2. In conclusion, test item SIKA Hardener MTJ applied to the rabbits' eye mucosa, caused slight to severe conjunctivae and slight cornea irritant effects, fully reversible within 1 week. According to Regulation (EC) No 1272/2008 (CLP), the test item has to be classified into “Category 2 (reversible effects on the eye/irritating to eyes)” on basis of cornea opacity.

Endpoint conclusion:

adverse effect observed (irritating)

Endpoint conclusion:

no study available

Skin corrosion in vitro

The purpose of the study was to determine the skin corrosion potential of the test item SIKA Hardener MTJ on reconstituted human epidermis in the EPISKIN model in vitro. Disks of EPISKIN (two units / chemical / incubation time) were treated with the test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in 5% CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue, viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 107 %. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. In conclusion, in the in vitro EPISKIN model test with SIKA Hardener MTJ the results indicated that the test item is not corrosive to skin.

Skin irritation in vitro

The purpose of the study was to determine the skin irritation potential of the test item SIKA Hardener MTJ on reconstituted human epidermis in the EPISKIN model in vitro.

Disks of epidermal units (three units / chemical) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically.

SDS 5 % aq. and 1 x PBS treated epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control.

The test item is considered to be a skin irritant, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less than or equal to (≤) 50% when compared to the viability values obtained from the negative control.

In this in vitro skin irritation test using the EPISKIN model, the test item SIKA Hardener MTJ did not show significantly reduced cell viability in comparison to the negative control. All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. According to the current OECD Guideline No. 439, SIKA Hardener MTJ is considered as non-irritant to skin and is therefore not classified.

Eye corrosion in vitro

The purpose of the Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item SIKA Hardener MTJ by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as ocular corrosive and/or severe irritant. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes.

In this ICET, SIKA Hardener MTJ did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. The experiment was considered to be valid.

In the In vitro eye corrosives and severe irritants study, using the Isolated Chicken Eyes model with SIKA Hardener MTJ, according to OECD guideline No. 438 ocular corrosion and severe irritation potential was not observed.

Eye Irritation Test in vivo

The acute eye irritation study of the test item SIKA Hardener MTJ was performed in three New Zealand White rabbits. The irritation effect of the test item was evaluated according to the Draize method (OECD No.: 405, 2012). The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A volume of 0.1 mL of the test item was used in pure state, as a single dose. The eyes of the test animals were not washed out after the application of test item. The eyes were examined at 1, 24, 48 and 72 hours, then 1 week after the application. One hour after the treatment, slight conjunctival redness, slight to moderate chemosis and moderate to severe discharge, as well as slight cornea opacity were observed. The area of cornea involved was greater than three quarters, up to the whole area in all animals. 72 hours after treatment slight redness and cornea opacity were recorded. The area of cornea involved was greater than three quarters, up to the whole area. 1 week after the treatment, all animals were free of symptoms, so the study was terminated. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows:

cornea opacity: 0.33, 1.00, 1.00

iris: 0.00, 0.00, 0.00

erythema: 1.00, 1.00, 1.00

chemosis: 0.33, 0.66, 0.33

discharge: 0.00, 1.33, 0.66

No systemic toxicity was observed on the day of the treatment and during the 1-week observation period. The body weight of animals corresponded to their species and age. Sign of pain and distress as discharge was observed in all animals. These symptoms were detected between treatment day and Day 2. In conclusion, test item SIKA Hardener MTJ applied to the rabbits' eye mucosa, caused slight to severe conjunctivae and slight cornea irritant effects, fully reversible within 1 week.

Justification for selection of skin irritation / corrosion endpoint:
Both a skin corrosion study and a skin irritation study in vitro according to OECD/EU guideline are available. The skin irritation study revealed that the test item is not irritating to the skin.

Justification for selection of eye irritation endpoint:
One in vivo Acute Eye Irritation GLP study according to OECD/EU guidelines available.

Effects on eye irritation: irritating

Skin irritation

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for skin irritation under Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.

Eye irritation

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered to be classified for eye irritation cat. 2, H319 under Regulation (EC) No 1272/2008, as amended for the fifteenth time in Regulation (EU) No 2020/1182.