N-[3-({[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino}methyl)-3,5,5-trimethylcyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

Endpoint summary

Description of key information

The test item Sika Hardener MI was tested for skin irritation/corrosion potential by in vitro Human Skin Model Tests. In the test for skin corrosion potential according to EU method B.40-bis and OECD 431, the test item was not considered to show skin corrosion potential. In the test for skin irritation potential according to EU method B.46 and OECD 439, it was concluded that the test item Sika Hardener MI shows skin irritation potential.

The test item Sika Hardener MI was tested for eye irritation/corrosion potential in the Acute Eye Irritation Study according to EU method B.5 and OECD 405. Applied to the rabbits' eye, the test item caused slight to severe conjunctivae and slight to moderate cornea irritant effects, fully reversible within 1 week.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2011-04-06 to 2011-05-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EU-Method B.40 BIS. „IN VITRO SKIN CORROSION: HUMAN SKIN MODEL TEST“ , 31 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

OECD 431; 13 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL of the liquid test item were applied to each tissue and spread to match the tissue size.

Duration of treatment / exposure:

3 minutes and 1 hours.

Remarks on result:

no indication of irritation

Remarks:

Sika Hardener MI was not considered corrosive in the Human Skin Model Test.

Other effects / acceptance of results:

After three minutes treatment with the test item, the relative absorbance values were reduced to 82.3 %. This value is well above the threshold for corrosion potential (50 %). After one hour treatment, relative absorbance values were reduced to 42.5 %. This value, too, is well above the threshold for corrosion potential (15 %). Therefore, Sika Hardener MI was not considered corrosive in the Human Skin Model Test.

Interpretation of results:

GHS criteria not met

Conclusions:

Sika Hardener MI was not considered corrosive in the Human Skin Model Test.

Executive summary:

The determination of Skin Corrosion Potential of Sika Hardener MI was performed in the Human Skin Model Test following OECD Guideline 431 and EU Method B.40-BIS. One valid experiment was performed. Two tissues of the human skin model EpiDermTM were treated with Sika Hardener MI for three minutes and one hour, respectively. 50 µL of the liquid test item were applied to each tissue and spread to match the tissue size. Deionised water was used as negative control, 8 M KOH was used as positive control.

After three minutes treatment with the test item, the relative absorbance values were reduced to 82.3 %. This value is well above the threshold for corrosion potential (50 %). After one hour treatment, relative absorbance values were reduced to 42.5 %. This value, too, is above the threshold for corrosion potential (15 %). Therefore, Sika Hardener MI was not considered corrosive in the Human Skin Model Test.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2011-08-11 to 2011-10-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 761/2009, Method B.46, adopted 23 July 2009: “In Vitro Skin Irritation: Reconstructed human epidermis model test”

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Version 439, 22 July 2010, “In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size (0.63 cm²; as indicated by supplier).

Remarks on result:

positive indication of irritation

Remarks:

Sika Hardener MI is considered as “irritant in the Human Skin Model Test”.

Other effects / acceptance of results:

After the treatment with the test item, the relative absorbance values were reduced to 5.8 %. This value is well below the threshold for irritation potential (50%). Therefore, Sika Hardener MI is considered as “irritant in the Human Skin Model Test”.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Sika Hardener MI is considered as “irritant in the Human Skin Model Test”.

Executive summary:

The determination of the skin irritation potential of Sika Hardener MI was performed in the Human Skin Model Test following Commission Regulation (EC) No. 761/2009, Method B.46, adopted 23 July 2009: “In Vitro Skin Irritation: Reconstructed human epidermis model test” and according to OECD Guideline for the Testing of Chemicals, Version 439, 22 July 2010, “In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method”. One valid experiment was performed. Three tissues of the human skin model EpiDerm^TMwere treated with Sika Hardener MI for 60 minutes. 30 µL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size (0.63 cm²; as indicated by supplier). DPBS-buffer was used as negative control, 5% SDS-solution was used as positive control. All validity criteria were met. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18 %). After the treatment with the test item, the relative absorbance values were reduced to 5.8 %. This value is well below the threshold for irritation potential (50%). Therefore, Sika Hardener MI is considered as “irritant in the Human Skin Model Test”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2014-04-14 to 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 animals
- Sex: male
- Weight at study initiation: 2958 - 3200 g
- Housing: single housing in metal cages
- Diet: CRLT/ny rabbit diet (Szindbád Kft., 2100 Gödöllő, Szárítópuszta, Hungary) ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Humidity: 30 - 70 %
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL

Duration of treatment / exposure:

The test substance was applied once to each animal (single treatment). 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.

Observation period (in vivo):

The eyes were examined at 1, 24, 48 and 72 hours, then 1 week after the application. The duration of the observation period was sufficient for the statement of reversibility or irreversibility of changes. Any clinical signs of toxicity or signs of pain and/or distress were recorded twice daily, with a minimum of 6 hours between observations.

Number of animals or in vitro replicates:

3 males

Details on study design:

REMOVAL OF TEST SUBSTANCE
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 (2012-10-02).

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:

0.66

Max. score:

3

Reversibility:

fully reversible within: 72 h

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 48 h

Irritation parameter:

chemosis score

Basis:

animal #3

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 #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 1 week

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:

0.66

Max. score:

4

Reversibility:

fully reversible within: 72 h

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

Other effects:

Signs of pain and distress as discharge were observed in all animals. These symptoms were detected between treatment day and day 2.

Irritant/corrosive response data for each animal at each observation time up to removal of each animal from the test

Score at time point / Reversibility	Cornea	Iris	Conjunctivae	Chemosis
	Max. score: 4	Max. score: 2	Max. score: 3	Max. score: 4
60 min	2, 1, 1	0, 0, 0	1, 1, 1	1, 1, 1
24 h	2, 1, 1	0, 0, 0	1, 1, 1	1, 1, 1
48 h	2, 1, 1	0, 0, 0	1, 1, 1	0, 0, 0
72 h	2, 1, 0	0, 0, 0	1, 1, 0	0, 0, 0
Average 24h, 48h, 72h	2.00, 1.00, 0.66	0, 0, 0	1.00, 1.00, 0.66	0.33, 0.33, 0.33
Reversibility*)	c.	-	c.	c.
Average time for reversion	72 h - 1 week	-	1 week	48 h

 

*) Reversibility: c. = completely reversible; n.c. = not completely reversible; n. = not reversible

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The test item Sika Hardener MI applied to the rabbits' eye mucosa caused slight to severe conjunctivae and slight to moderate cornea irritant effects, fully reversible within 1 week.

Executive summary:

The acute eye irritation study of the test item Sika Hardener MI 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 and chemosis, moderate to severe discharge, as well as slight to moderate 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 slight to moderate cornea opacity were recorded. The area of cornea involved was greater than three quarters, up to the whole area. One animal became free of symptoms by this time point.

The individual scores (mean over the time points 24, 48 and 72 hours) for animal 1 were determined as: conjunctivae: 1, chemosis: 0.33, cornea: 2 and iris: 0. For animal 2 the following scores were determined: conjunctivae: 0.66, chemosis: 0.33, cornea: 1, iris:0, and for animal 3: conjunctivae: 1, chemosis: 0.33, cornea: 0.66 and iris: 0.

1 week after the treatment, all animals were free of symptoms, so the study was terminated. 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. Signs of pain and distress as discharge were observed in all animals. These symptoms were detected between treatment day and day 2.

The test item Sika Hardener MI applied to the rabbits' eye mucosa caused slight to severe conjunctivae and slight to moderate cornea irritant effects, fully reversible within 1 week.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2011-08-17 to 2011-09-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

OECD Guideline for the Testing of Chemicals, Method No. 437, edition adopted 07 Sept. 2009: “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

Cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour.

Vehicle:

other: olive oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

10 % solution in olive oil

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Remarks on result:

no indication of irritation

Remarks:

The test item Sika Hardener MI showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 2.2640.

Other effects / acceptance of results:

The test item Sika Hardener MI was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without Phenol red at 32 ± 1 °C for one hour and whose opacity had been determined. The test item was incubated on the cornea for 10 minutes/four hours at 32 ± 1 °C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured.
Physiological sodium chloride solution was used as negative control, sodium hydroxide (10 % solution in 0.9 % NaCl solution) was used as positive control. The negative control showed no irritation. The positive control induced a very severe irritation on the cornea.
The test item was tested pure. A mean IVIS of 2.2640 was calculated, corresponding to a ICCVAM classification as mild eye irritant.

Interpretation of results:

other: not severely irritating

Conclusions:

The test item Sika Hardener MI showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 2.2640.
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sika Hardener MI possesses no severe eye irritation potential.

Executive summary:

The eye irritation/corrosion potential of the test item SIKA Hardener MI was evaluated in the BCOP Test following OECD 437 (September 2009). One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old. The test item Sika Hardener MI (tested as 10 % solution in olive oil) was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour and whose opacity had been measured. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured.

Physiological sodium chloride solution was used as negative control. Olive oil was used as solvent control. The negative control and the solvent control showed no irritating effect on the cornea. 10 % sodium hydroxide solution was used as positive control. The positive control induced a very severe irritation on the cornea.

The test item Sika Hardener MI showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 2.2640.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sika Hardener MI possesses no severe eye irritation potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion:

The determination of Skin Corrosion Potential of Sika Hardener MI was performed in the Human Skin Model Test following OECD Guideline 431 and EU Method B.40-BIS. One valid experiment was performed. Two tissues of the human skin model EpiDermTM were treated with Sika Hardener MI for three minutes and one hour, respectively. 50 µL of the liquid test item were applied to each tissue and spread to match the tissue size. Deionised water was used as negative control, 8-m KOH was used as positive control.

After three minutes treatment with the test item, the relative absorbance values were reduced to 82.3 %. This value is well above the threshold for corrosion potential (50 %). After one hour treatment, relative absorbance values were reduced to 42.5 %. This value, too, is above the threshold for corrosion potential (15 %). Therefore, Sika Hardener MI was not considered corrosive in the Human Skin Model Test (LAUS, 2011).

 

The determination of the skin irritation potential of Sika Hardener MI was performed in the Human Skin Model Test following Commission Regulation (EC) No. 761/2009, Method B.46, adopted 23 July 2009: “In Vitro Skin Irritation: Reconstructed human epidermis model test” and according to OECD Guideline for the Testing of Chemicals, Version 439, 22 July 2010, “In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method”. One valid experiment was performed. Three tissues of the human skin model EpiDermTM were treated with Sika Hardener MI for 60 minutes. 30 µL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size (0.63 cm²; as indicated by supplier). DPBS-buffer was used as negative control, 5% SDS-solution was used as positive control. All validity criteria were met. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18 %). After the treatment with the test item, the relative absorbance values were reduced to 5.8 %. This value is well below the threshold for irritation potential (50%). Therefore, Sika Hardener MI is considered as “irritant in the Human Skin Model Test" (LAUS, 2011).

 

Eye irritation:

The eye irritation/corrosion potential of the test item SIKA Hardener MI was evaluated in the BCOP Test following OECD 437 (September 2009) and in the Acute Eye Irritation Study according to EU method B.5 and OECD 405.

 

BCOP Test:

One valid experiment was performed. Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old. The test item Sika Hardener MI (tested as10 % solution in olive oil) was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for one hour and whose opacity had been measured. The test item was incubated on the cornea for 10 minutes at 32 ± 1 °C. After removal of the test item and two hours post-incubation, opacity and permeability values were measured.

Physiological sodium chloride solution was used as negative control. Olive oil was used as solvent control. The negative control and the solvent control showed no irritating effect on the cornea. 10 % sodium hydroxide solution was used as positive control. The positive control induced a very severe irritation on the cornea.

The test item Sika Hardener MI showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 2.2640.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item Sika Hardener MI possesses no severe eye irritation potential (LAUS, 2011).

 

Acute Eye Irritation Study in rabbits:

On the basis of the result of the BCOP Test an in vivo study according to according to EU method B.5 and OECD Guideline 405 could further be conducted which should enable classification and labelling as an eye damage category 2 according to Regulation (EC) No 1272/2008 (CLP). Three male New Zealand White rabbits were used in the test. 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 and chemosis, moderate to severe discharge, as well as slight to moderate cornea opacity were observed. The area of cornea involved was greater than three quarters, up to the whole area in all animals.

The individual scores (mean over the time points 24, 48 and 72 hours) for animal 1 were determined as: conjunctivae: 1, chemosis: 0.33, cornea: 2 and iris:0. For animal 2 the following scores were determined: conjunctivae: 0.66, chemosis: 0.33, cornea: 1, iris: 0, and for animal 3: conjunctivae: 1, chemosis: 0.33, cornea: 0.66 and iris: 0.

72 hours after treatment slight redness and slight to moderate cornea opacity were recorded.The area of cornea involved was greater than three quarters, up to the whole area. One animal became free of symptoms by this time point.

1 week after the treatment, all animals were free of symptoms, so the study was terminated.

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 MI applied to the rabbits' eye mucosa, caused slight to severe conjunctivae and slight to moderate cornea irritant effects, fully reversible within 1 week. The test item should be classified as category 2 according to Regulation (EC) No 1272/2008 (CLP).

Justification for classification or non-classification

Based on the results of skin irritation studies, Sika Hardener MI is classified and labelled as skin irritant Categorgy 2 according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No. 2017/776.

 

The results of the in vivo eye irritation study lead to classification of the test item into Category 2 (H319, causes serious eye irritation) according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in regulation (EC) No. 2017/776.