Hexane, 1,6-diisocyanato-, homopolymer, 2-hydroxyethyl acrylate- and propylene glycol monoacrylate-blocked

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 13, 2016 to May 11, 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured to form a multi-layered, highly differentiated model of the human epidermis

Source strain:

not specified

Justification for test system used:

Validated in vitro model

Vehicle:

unchanged (no vehicle)

Details on test system:

- The test system is a commercially available EpiDermTM-Kit, procured by MatTek. The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.
- Viability: assessed by the MTT test

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test substance (pure) was applied directly to each tissue and spread to match the tissue size (0.63 cm2)

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3 replicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

MTT test

Value:

2.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the study conditions, the substance was considered to be irritating to skin in the reconstructed human epidermis (RHE) test method.

Executive summary:

A study was conducted to determine the skin irritation potential of the substance according to OECD Guideline 439 (reconstructed human epidermis (RHE) test method), in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated with the test substance for 60 min. The test substance was applied to each tissue and spread to match the tissue size (0.63 cm2). The post incubation period was in total 42 h (at 37 ± 1°C and 5.0 ± 0.5% CO2). DPBS buffer was used as negative control, and 5% SDS solution as positive control. After treatment, the test and the control substance were rinsed off from the tissues. Cell viability of the tissues was then evaluated by the addition of MTT. Viability was evaluated by the enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured (optical density) after extraction from tissues. Values for the negative and positive controls were within the range of historical data, hence the experiment was considered valid. Following exposure with the test substance, cell viability was reduced to 2.5%. Under the study conditions, the substance was considered to be irritating to skin (Andres, 2017).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed human skin model

Justification for test system used:

Validated in vitro model

Vehicle:

unchanged (no vehicle)

Details on test system:

- The test system is a commercially available EpiDerm-Kit, procured by MatTek. The EpiDerm tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues are cultured on specially prepared cell cultures inserts.
- Origin: EpiDerm tissues were procured from MatTek in vitro Life Science Laboratories.
- Viability: assessed by the MTT test

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The liquid test substance was applied without preparation (50 μL)

Duration of treatment / exposure:

Incubation time: 0 3 min at room temperature and 1 h at 37 ± 1°C and 5.0 ± 0.5% CO2

Duration of post-treatment incubation (if applicable):

-

Number of replicates:

6 in each of the two tests

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min exposure

Run / experiment:

MTT test

Value:

97.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Remarks:

1 hour exposure

Run / experiment:

MTT test

Value:

89.6

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:

- The mean relative absorbance value was reduced to 97.4% after 3 min treatment with the test substance. This value is above the threshold indicating corrosivity (50%). After 1 h treatment, the mean relative absorbance value was reduced to 89.6%, lying above the threshold for corrosivity (15%) as well. Therefore, the test substance is considered as non-corrosive to skin.
- The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: mean optical density values were 1.9 (3 min) and 1.8 (1 h). The positive control showed clear corrosive effects. The criterion for the viability of the 1 h experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The viability was 8.0%. Values for negative control and for positive control were within the range of historical data of the test facility. Therefore the experiment is considered valid.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the substance was considered to be non-corrosive to skin in the reconstructed human epidermis (RHE) test method.

Executive summary:

A study was conducted to determine the skin corrosion potential of the substance according to OECD Guideline 431 (reconstructed human epidermis (RHE) test method), in compliance with GLP. Two tissues of the human skin model EpiDerm were treated with the test substance for 3 min and 1 h, respectively. The test substance (without vehicle) was applied to each tissue and spread to match the tissue size. Demineralised water was used as negative control, and KOH as positive control. After treatment, the test substance and the control substances were rinsed off from the tissues. Cell viability was then evaluated by the addition of MTT (which allows the measurement of the enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively analysed (optical density) after extraction from tissues). After 3 min of exposure, the mean relative absorbance values obtained with the test substance were 97.4% compared to the negative control. This value was well above the threshold of 50%. After 1 h of treatment, the mean relative absorbance value was 89.6%. This value was also above the threshold. After treatment with the negative control, the absorbance values were within the required acceptability criterion. The positive control showed clear corrosive effects for both treatment intervals. The experiment was considered valid. Under the study conditions, the substance was considered to be non-corrosive to skin (Andres, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Bos primigenius Taurus

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s Balanced Salt Solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1°C in an incubation chamber for 1 h.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance (pure) was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test substance.

Duration of treatment / exposure:

10 min at 32 ± 1°C

Observation period (in vivo):

-

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1°C

Number of animals or in vitro replicates:

For each treatment group (negative control solution, test substance and positive control), three replicates were used.

Details on study design:

- The BCOP (The Bovine Corneal Opacity and Permeability) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability.
- After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage, therefore, all corneas were used. For each treatment group (negative control solution, test substance and positive control), three replicates were used. After removal of the pre-incubation medium, negative control and positive control solution were applied by pipetting 750 μL of the appropriate liquid through the refill hole in the holder on the cornea. In order to apply the test substance, the nut was unscrewed to remove the glass disc. Then, the test substance was applied on the epithelium in such a manner that as much as possible of the cornea was covered with the test substance. Exposure time on the corneas was 10 min at 32 ± 1°C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 h at 32 ± 1°C (post-incubation). After the post-incubation, the cMEM without phenol red was renewed in both chambers. Then, the final opacity value of each cornea was recorded. The cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 4 mg/mL) was added to the front chamber. The chambers were closed again and incubated for 85 min at 32 ± 1°C. After incubation, the content of the posterior chamber was thoroughly mixed. Finally, the corneal permeability was measured with the spectral photometer at 492 nm.

Irritation parameter:

in vitro irritation score

Remarks:

corneal opacity and permeability

Run / experiment:

BCOP test

Value:

4.41

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

In the negative control, no signs of eye irritation were observed. The positive control induced serious eye damage. The test substance showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 4.41. The experiment is considered as sufficient for a reliable calculation of the IVIS of the test substance, because all three replicates of the test substance led to the same assessment for the test substance (IVIS for each replicate of the test substance: 5.70; 3.25; 4.28; Mean IVIS: 4.41; Relative Standard Deviation IVIS: 27.98%).

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the study conditions, no prediction for eye irritation can be made.

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability test method), in compliance with GLP. Bovine corneas were collected from cattle. One valid experiment was performed with three replicates each for the negative control, the positive control (dimethyl formamide) and the test substance. The pure, undiluted test substance was applied for 10 min at 32°C in a way that as much as possible of the cornea surface was covered. After removal of the test substance and a 2 h post-incubation, opacity and permeability values were measured to give the in vitro irritation score (IVIS). The IVIS for the test substance was determined to be 4.41 (i.e. between > 3 and ≤ 55). No signs of eye irritation were observed following exposure with the negative control. The positive control induced serious eye damage. Both the controls met the validity criteria. Under the study conditions, the test substance produced a in vitro irritancy score of 4.41 and therefore, according to OECD Guideline 437 criteria, no prediction for eye irritation can be made (Andres, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation- in vitro (OECD 439):

A study was conducted to determine the skin irritation potential of the substance according to OECD Guideline 439 (reconstructed human epidermis (RHE) test method), in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated with the test substance for 60 min. The test substance was applied to each tissue and spread to match the tissue size (0.63 cm2). The post incubation period was in total 42 h (at 37 ± 1°C and 5.0 ± 0.5% CO2). DPBS buffer was used as negative control, and 5% SDS solution as positive control. After treatment, the test substance and the control substance were rinsed off from the tissues. Cell viability of the tissues was then evaluated by the addition of MTT. Viability was evaluated by the enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured (optical density) after extraction from tissues. Values for the negative and positive controls were within the range of historical data, hence the experiment was considered valid. Following exposure with the test substance, cell viability was reduced to 2.5%. Under the study conditions, the substance was considered to be irritant to skin (Andres, 2017).

Skin corrosion - in vitro (OECD 431):

A study was conducted to determine the skin corrosion potential of the substance according to OECD Guideline 431 (reconstructed human epidermis (RHE) test method), in compliance with GLP. Two tissues of the human skin model EpiDerm were treated with the test substance for 3 min and 1 h, respectively. The test substance (without vehicle) was applied to each tissue and spread to match the tissue size. Demineralised water was used as negative control, and KOH as positive control. After treatment, the test and the control substances were rinsed off from the tissues. Cell viability was then evaluated by the addition of MTT (which allows the measurement of the enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively analysed (optical density) after extraction from tissues). After 3 min of exposure, the mean relative absorbance values obtained with the test substance were 97.4% compared to the negative control. This value was well above the threshold of 50%. After 1 h of treatment, the mean relative absorbance value was 89.6%. This value was also above the threshold. After treatment with the negative control, the absorbance values were within the required acceptability criterion. The positive control showed clear corrosive effects for both treatment intervals. The experiment was considered valid. Under the study conditions, the substance was considered to be non-corrosive to skin (Andres, 2017).

Eye irritation - in vitro (OECD 437):

A study was conducted to determine the eye irritation potential of the substance according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability test method), in compliance with GLP. Bovine corneas were collected from cattle. One valid experiment was performed with three replicates each for the negative control, the positive control (dimethyl formamide) and the test substance. The pure, undiluted test substance was applied for 10 min at 32°C in a way that as much as possible of the cornea surface was covered. After removal of the test substance and a 2 h post-incubation, opacity and permeability values were measured to give the in vitro irritation score (IVIS). The IVIS for the test substance was determined to be 4.41 (i.e. between > 3 and ≤ 55). No signs of eye irritation were observed following exposure with the negative control. The positive control induced serious eye damage. Both the controls met the validity criteria. Under the study conditions, the substance produced a in vitro irritancy score of 4.41 and therefore, according to OECD Guideline 437 criteria, no prediction for eye irritation can be made (Andres, 2017).

Justification for classification or non-classification

The substance did not show corrosive effects in an in vitro skin corrosion study (OECD Guideline 431), however it was considered to be irritating to skin in an in vitro skin irritation study (OECD Guideline 439). Hence, the substance warrants Skin Irrit. 2 - H315 (causes skin irritation) classification according to EU CLP (EC 1272/2008) criteria.

The substance showed some effects on the cornea (opacity and permeability) of the bovine eye in anin vitroeye irritation study, but not sufficient to be classified as inducing serious eye damage. In accordance with the “Guide to the classification and labelling of UV/EB acrylates” proposed by the UV/EB Acrylate Resins Sector Group (https://hnlkg4f5wdw34kx1a1e9ygem-wpengine.netdna-ssl.com/wp-content/uploads/2017/09/4_Guide-to-the-Classification-and-Labelling-of-UV-EB-Acrylates.pdf), acrylated substances are classified as Category 2 skin and eye irritants by default unless there is data available that would lead to a different classification. Therefore, based on the above-mentioned guidance, the substance warrants classification asEye Irrit. 2 – H319 (causes serious eye irritation).