Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate

Endpoint summary

Description of key information

Skin corrosion (OECD 431): not corrosive

Skin irritation (OECD 439): not irritating

Eye irritation (OECD 437): not irritating

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:

30 Mar - 01 Apr 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted in 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted in 2008

Qualifier:

according to guideline

Guideline:

other: ECVAM INVITTOX Protocol No 118: "EpiSkinTM Skin Corrosivity Test"

Version / remarks:

adopted in 2011

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test system:

human skin model

Source species:

human

Cell type:

other: normal human epidermal keratinocytes (NHEK)

Source strain:

other: reconstructed three-dimensional human epidermis model (SkinEthic)

Justification for test system used:

This test uses the EPISKIN-SMTM reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. This method allows the identification of corrosive and non-corrosive substances and mixtures in accordance with UN GHS. It further allows a partial sub-caregorisation of corrosives in optional sub-category 1A or a combination of optional sub-categories 1B and 1C.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EPISKIN-SMTM (SkinEthic) consisting of normal human epidermal keratinocytes
- Tissue batch number: 16-EKIN-013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL, about 15 times

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 15 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
For historical control data see table 1.

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin in accordance with Regulation EC 1272/2008 and UN GHS "Category 1" if the relative tissue viability after 4 h treatment is decreased below 35%: if viability is reduced to less than 35% after 4 h treatment and to more than 35% after 60 min treatment or to less than 35% viability after 60 min but not more than 35% after 3 min treatment, the test item is classified as corrosive, in accordance with optional UN GHS sub-categories 1B and 1C. A test item which decreases viability below 35% after 3 min treatment is classified as corrosive in accordance with optional UN GHS sub-category 1A.
- The test substance may be considered as non-corrosive to skin in accordance with UN GHS "No Category" if realtive tissue viability after 4 h treatment is not decreased to less than 35% of the corresponding negative control tissues.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 ± 2 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 0.9%

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 100%

Duration of treatment / exposure:

4 h, 60 min and 3 min

Duration of post-treatment incubation (if applicable):

Not applicable

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

59

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: no indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not examined

Remarks on result:

other: no indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

4 h

Value:

58

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

2% tissue viability

Remarks on result:

other: no indication of corrosion

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: A mixture of 20 mg test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn purple/blue. Therefore, the non-specific reduction of MTT equaled 0%.
- Colour interference with MTT: A mixture of 10 mg of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore, non-specific colour of additional viable tissues equaled 0%.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The controls confirmed the validity of the study.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: See table 1 for historical control data.

Interpretation of results:

other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/208

Conclusions:

CLP: not classified

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jun - 01 Jul 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2012

Qualifier:

according to guideline

Guideline:

other: ECVAM DB-ALM Protocol No. 131 "EpiSkinTM Skin Irritation Test (15 min - 42 hours)

Version / remarks:

adopted in 2012

Qualifier:

according to guideline

Guideline:

other: LÓréal Standard Operating Procedure "EpiSkinTM Skin Irritation Test Method"; - ECVAM Skin Irritation Validation Study - Validation of the EpiSkinTM Test Method (15 min - 42 hours) for the Prediction of Acute Skin Irritation of Chemicals, Version 1.8

Version / remarks:

adopted in 2009

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Test system:

human skin model

Source species:

human

Cell type:

other: normal human epidermal keratinocytes (NHEK)

Source strain:

other: reconstructed three-dimensional human epidermis model (SkinEthic)

Justification for test system used:

This test uses the EPISKIN-SMTM reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS "Category 2". Depending on the regulatory framework it can also be used to identify non-classified chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EPISKIN-SMTM (SkinEthic) consisting of normal human epidermal keratinocytes
- Tissue batch number: 16-EKIN-026

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1°C
- Temperature of post-treatment incubation: 37 ± 1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
Tissues were washed with Dulbecco´s phosphate buffered saline (DPBS) to remove any residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter: band pass filter
- Filter bandwidth: 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
For historical control data see table 1.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin in accordance with Regulation EC 1272/2008 and UN GHS "Category 2" if the viability after 15 min of exposure and 42 h of post-incubation is less than or equal to 50%.
- The test substance may be considered as non-irritant to skin in accordance with UN GHS "No Category" if the tissue viability after exposure and post-treatment incubation is higher than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg test substance + 5 µL DPBS

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 ± 0.5 min

Duration of post-treatment incubation (if applicable):

42 ± 1 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

6.2% tissue viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: A mixture of 10 mg test item per 2 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn purple/blue. Therefore, the non-specific reduction of MTT equaled 0%.
- Colour interference with MTT: The mixtures of 10 mg of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, non-specific colour of additional viable tissues equaled 0%.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The controls confirmed the validity of the study.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: See table 1 for historical control data.

Interpretation of results:

other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/208

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jul 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted in 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: Fresh eyes were transported in Hanks´balanced salt solution containing penicillin/streptomycin on ice. Immediately after arrival of the eyes at the laboratory, cornea preparation was initiated.
- Indication of any existing defects or lesions in ocular tissue samples: no (visual examination)
- Indication of any antibiotics used: no

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 100%

Duration of treatment / exposure:

10 min at 32 ± 1°C

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1°C

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were carefully visually examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled way and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS and mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers were filled with RPMI medium (without phenol red) containing 1% fetal bovine serum and 2 mM L-glutamine (complete RPMI). The corneas were equilibrated for 1 h at at 32 ± 1°C. After the equilibration period, the medium was replaced with fresh complete RPMI.

QUALITY CHECK OF THE ISOLATED CORNEAS
An initial measurement was performed on each of the corneas using the opacitometer.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
0.9% NaCl (physiological saline)

POSITIVE CONTROL USED
100% ethanol

APPLICATION DOSE AND EXPOSURE TIME
750 µL of the test substances and control substances for 10 min at 32 ± 1°C

TREATMENT METHOD: open chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times

- POST-EXPOSURE INCUBATION: 2 h at 32 ± 1°C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: illuminance measurement using a calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria as indicated in the TG was used

Irritation parameter:

in vitro irritation score

Value:

-0.06

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: IVIS of the positive control (100% ethanol; 32 replicates): 46.61 (mean value, MV); 9.15 (standard deviation, SD); 28.3 (MV - 2 x SD); 64.92 (MV + 2 x SD)

Irritant / corrosive response data:

With the negative control (0.9% (w/v) NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging. Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score (IVIS) was -0.06.

Interpretation of results:

other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reliable studies regarding skin irritation/ corrosion and eye irritation are available for the test substance.

Skin:

The acute skin corrosion potential of the test substance was assessed in an in vitro study performed according to OECD Guideline 431 and in compliance with GLP (Lehmeier, 2016). Cytotoxic effects of the test substance on a reconstituted three-dimensional human epidermis model (EPISKIN-SMTM) consisting of normal human epidermal keratinocytes following topical application were determined. Tissue viability was evaluated via the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min, 60 min and 4 h exposure period compared to the negative controls. Glacial acetic acid was used as positive control and 0.9% NaCl solution served as negative control. Relative viability was determined to 59% after 3 min, 102% after 60 min and 58% after 4 h treatment. The relative mean tissue viability was not decreased to less than 35%. Thus, under the conditions of this study, the test substance is considered to be non-corrosive to the skin.

The acute skin irritation potential of the test substance was assessed in an in vitro study performed according to OECD Guideline 439 and in compliance with GLP (Lehmeier, 2016). Cytotoxic effects of the test substance on a reconstituted three-dimensional human epidermis model (EPISKIN-SMTM) consisting of normal human epidermal keratinocytes following topical application were determined. Tissue viability was evaluated via the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 15 min exposure period and 42 h post-incubation period compared to the negative controls. Sodium dodecyl sulfate (5%) was used as positive control and Dulbecco´s phosphate buffered saline served as negative control. Relative mean viability was determined to 96.5%. Thus, under the conditions of this study, the test substance is considered to be non-irritant to the skin.

Eye:

The acute eye irritation potential of the test substance was assessed in an in vitro study performed according to OECD Guideline 437 and in compliance with GLP (Bachmann, 2016). In the BCOP test pre-incubated isolated corneas were exposed to the test substance for 10 min followed by a 2 h post-incubation period. Opacity and permeability of the corneas were determined. Ethanol (100%) was used as positive control and 0.9% NaCl solution served as negative control. With a mean in vitro irritation score of -0.06 the test substance showed no effect on the cornea of the bovine eye. Thus, under the conditions of this study, the test substance is considered to be non-irritant to the eye.

Justification for classification or non-classification

The available data on skin corrosion and irritation and eye irritation do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.