Hexamethylene diisocyanate, oligomers, reaction products with 4-Hydroxybutyl acrylate

Endpoint summary

Description of key information

Skin:

in-vitro skin corrosion: not corrosive

in-vitro skin irritation: irritating Cat. 2

Eye:

in-vitro Eye irritation: 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:

2017

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16/0438-1, Batch 360-71-3
- The test substance was homogenous by visual inspection.
- pH 5

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: undiluted test substance moistened with deionized water

FORM AS APPLIED IN THE TEST (if different from that of starting material): moistened with water

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Details on animal used as source of test system:

- Tissue model: EPI-200
- Tissue Lot Number: 23388
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Justification for test system used:

The test system is part of an integrated approach on testing and assessment (IATA) for skin corrosion and irritation and is one of the accepted guideline studies.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm model
- Tissue batch number(s): 23388

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C and room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

NUMBER OF REPLICATE TISSUES: 2 tissues per exposure time

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 45%.
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is higher than 45%, or if the viability after 3 minutes exposure is greater than or equal to 45 % and the viability after 1 hour exposure is less than 10%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than 55% and the viability after 1 hour exposure is greater than 20%.
- The borderline evaluation was determined statistically using historic data and hence considers the variance of the test method.This evaluation is an amendment to the evaluation provided in OECD Guideline 431.

Control samples:

yes, concurrent vehicle

other: Positive control: 8-N potassium hydroxide solution

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Fifty microliters (50 µL) undiluted, at ca. 37 °C heated test substance were applied by using a pipette.

Duration of treatment / exposure:

3 minutes at room temperature and 1h in the incubator

Duration of post-treatment incubation (if applicable):

Rinsed tisuues were kept in 24-well plates at room temperature on assay medium until all tissues per application time were dosed and rinsed.

Number of replicates:

2 tissues per exposure time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (Exposure period. 3min)

Value:

83.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (Exposure period: 1h)

Value:

92.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Exposure period 3 minutes: Individual and mean OD570 values, individual and mean viability values and standard deviations

Test substance		Tissue 1	Tissue 2	Mean	SD
NC	Mean OD570	1.527	1.721	1.624
NC	Viability (% of NC)	94.0	106.0	100.0	8.5
Test substance	Mean OD570	1.249	1.475	1.362
Test substance	Viability (% of NC)	76.9	90.8	83.9	9.8
PC	Mean OD570	0.159	0.244	0.202
PC	Viability (% of NC)	9.8	15.0	12.4	3.7

Exposure period 1h: Individual and mean OD570 values, individual and mean viability values and standard deviations

Test substance		Tissue 1	Tissue 2	Mean	SD
NC	Mean OD570	1.659	1.564	1.612
NC	Viability (% of NC)	102.9	97.1	100.0	4.1
Test substance	Mean OD570	1.348	1.625	1.487
Test substance	Viability (% of NC)	83.6	100.9	92.2	12.2
PC	Mean OD570	0.097	0.085	0.091
PC	Viability (% of NC)	6.0	5.3	5.6	0.5

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed and by applying the evaluation criteria it was concluded that the test substance does not show a skin corrosion potential under the test conditions chosen.

Executive summary:

The objective was to assess the skin irritation and corrosion potential of the test substance. By using the currently available methods a single in vitro assay is not sufficient to cover the full range of

skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

The potential of the test substance to cause dermal corrosion was assessed by a single topical application of 50 μL of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).

For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. Tissue destruction was determined by measuring the metabolic activity of the tissue after

exposure by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The final mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 83.9% and 92.2% after an exposure period of 1 hour.

Based on the results observed and by applying the evaluation criteria it was concluded that the test substance does not show a skin corrosion potential under the test conditions chosen.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16/0438-1, Batch 360-71-3
- The test substance was homogenous by visual inspection.
- pH 5

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: undiluted test substance moistened with deionized water

FORM AS APPLIED IN THE TEST (if different from that of starting material): moistened with water

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Details on animal used as source of test system:

- Tissue model: EPI-200
- Tissue Lot Number: 23388
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Justification for test system used:

The test system is part of an integrated approach on testing and assessment (IATA) for skin corrosion and irritation and is one of the accepted guideline studies.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm model
- Tissue batch number(s): 23388

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant if the viability is less than 45% compared to negative control.
- The test substance is considered to be non-irritant if the viability is higher than 55% compared to control.
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 45%, or if the viability after 3 minutes exposure is greater than or equal to 45 % and the viability after 1 hour exposure is less than 10%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than 55% and the viability after 1 hour exposure is greater than 20%.
- The borderline evaluation was determined statistically using historic data and hence considers the variance of the test method.This evaluation is an amendment to the evaluation provided in OECD Guideline 431.

Control samples:

other: Negative control: PBS; positive control: 5% SDS

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL undiluted, at 37°C heated test substance
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl sterile PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl SDS solution
- Concentration (if solution): 5%

Duration of treatment / exposure:

1h

Duration of post-treatment incubation (if applicable):

24h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Individual and mean OD570 values, individual and mean viability values and standard deviations

Test substance		Tissue 1	Tissue 2	Tissue 3	Mean	SD
NC	Mean OD570	1.900	1.797	1.810	1.836
NC	Viability (% of NC)	103.5	97.9	98.6	100.0	3.1
Test substance	Mean OD570	0.049	0.455	0.047	0.184
Test substance	Viability (% of NC)	2.7	24.8	2.6	10.0	12.8
PC	Mean OD570	0.036	0.041	0.036	0.037
PC	Viability (% of NC)	1.9	2.2	1.9	2.0	0.2

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Based on the results observed and by applying the evaluation criteria, it was concluded that the test item show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Executive summary:

The objective was to assess the skin irritation and corrosion potential of the test substance. By using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of 30µl of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).

The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour, followed by a 42-hour post-incubation period.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The final mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 10%.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

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

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16/438-1, 360-71-3
- pH 5
- The test substance was homogenous by visual inspection.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Source: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue model: OCL-200
- Tissue Lot Number: 23760
- Incubation conditions: 37°C +/- 1°C, 5% +/- 1% CO2, 90% +/- 5% relative humidity
- Detection agent: MTT
- Ectracting agent: Isopropanol
- Wash buffer: PBS

Controls:

other: Negative control: deionized sterile water; positive control: Neat methyl acetate

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µl undiluted test substance

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

2h

Number of animals or in vitro replicates:

2

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed with sterile PBS
- Time after start of exposure: 30 minutes

SCORING SYSTEM:
Mean tissue viability
(% of negative control) Prediction

< 55 Irritant
55 - 65 Borderline
> 65 Non-irritant

Irritation parameter:

other: Tissue viability (% of NC)

Run / experiment:

Mean

Value:

93.8

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:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance		Tissue 1	Tissue 2	Mean	Inter-tissue variability (%)
NC	Mean OD570	1.624	1.625	1.624
NC	Viability (% of NC)	100.0	100.0	100.0	0.1
Test item	Mean OD570	1.545	1.502	1.523
Test item	Viability (% of NC)	95.1	92.5	93.8	2.6
PC	Mean OD570	0.333	0.448	0.390
PC	Viability (% of NC)	20.5	27.6	24.0	7.1

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed for the EpiOcular Test alone and by applying the evaluation criteria it was concluded that the test item does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Executive summary:

The objective was to assess the eye irritating potential of the test substance. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

However, in case of the test substance, the results derived with EpiOcular alone were sufficient for a final assessment. Therefore, further testing in BCOP was waived.

The potential of the test substance to cause ocular irritation was assessed by a single topical application of undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™). Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a

tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The following results were obtained in the EpiOcular™ eye irritation assay:

The test substance is not able to directly reduce MTT. The final mean viability of the tissues treated with the test substance was 93.8%.

Based on the results observed in the EpiOcular Test alone and by applying the evaluation criteria, it was concluded that the test substance does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

The objective was to assess the skin irritation and corrosion potential of the test substance. By using the currently available methods a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential.

Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of ca. 50 μL (corrosion test) or 30µl (irritation test) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).

For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. The irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour, followed by a 42-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The following results were obtained in the EpiDerm™ skin corrosion/irritation test:

The test substance is not able to directly reduce MTT.

Results of the Corrosion Test (SCT):

The final mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 83.9% and 92.2% after an exposure period of 1 hour.

Results of the Irritation Test (SIT):

The final mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 10%.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance shows a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.

Eye irritation:

The objective was to assess the eye irritating potential of the test substance. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.

However, in case of the test substance, the results derived with EpiOcular alone were sufficient for a final assessment. Therefore, further testing in BCOP was waived.

The potential of the test substance to cause ocular irritation was assessed by a single topical application of undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™). Two EpiOcular™ tissues were incubated with the test substance for 30 minutes followed by a 2-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a

tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability.

The following results were obtained in the EpiOcular™ eye irritation assay:

The test substance is not able to directly reduce MTT. The final mean viability of the tissues treated with the test substance was 93.8%.

Based on the results observed in the EpiOcular Test alone and by applying the evaluation criteria, it was concluded that the test substance does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.