Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 16, 2017 to January 20, 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No. 1152/2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

CAS No. 162492-07-1; EINECS No:. 500-740-9; Batch No.: 1591ZG-101; Purity >98 %; Appearance: homogeneous white powder

Test system:

human skin model

Remarks:

EpiDermTM tissues

Source species:

human

Cell type:

non-transformed keratinocytes

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.

Origin:
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.
Designation of the kit: EPI-212-SIT

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

One plate was used as negative control; each tissue was treated with 30 μL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate was used as positive control; each tissue was treated with 30 μL 5% SDS solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.

One plate was used for treatment with the test substance:
For each plate, 3 tissues were used. The tissues were wetted with 25 μL DPBS buffer before applying the test substance and spreading it to match the tissue size.
The following amounts were applied to the tissues:
Replicate 1: 25.3 mg
Replicate 2: 25.1 mg
Replicate 3: 25.6 mg

Duration of treatment / exposure:

1 h

Duration of post-treatment incubation (if applicable):

23 h and 30 min

Number of replicates:

3 replicates

Irritation / corrosion parameter:

% tissue viability

Value:

99.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Results:
% Tissue viability (tissue 1): 99.9%
% Tissue viability (tissue 2): 99.9%
% Tissue viability (tissue 3): 98.4%
% Tissue viability (mean) 99.4%

Tissue Viability

The photometric absorbance of the negative controls is considered as 100%. For each replicate of test substance and positive control, tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls:
% Tissue Viability = (OD replicate test substance resp. positive control x 100%) / OD negative controls

Acceptance of the results:

- The test substance is considered as non-irritant to skin.
- After the treatment, the relative absorbance values were reduced to 99.4%. This value is above the threshold for skin irritation (50%).
- The optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8.
- The positive control has met the acceptance criterion too, demonstrating the validity of the test system.
- Variation within replicates was within the accepted range for negative control, positive control and test substance (required: ≤ 18%).

Validity:
OD of negative control: 2.0 (Required: ≥ 0.8 and ≤ 2.8)
% Tissue viability of positive control SDS: 1.8% (Required: ≤ 20% of negative control)

SD of mean viability of the tissue replicates (%):
0.6% (negative control)
0.2% (positive control)
0.8% (test substance)
(Required: ≤ 18%)

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, after the treatment with the test substance, the relative absorbance values were reduced to 99.4 %. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance is considered as non-irritant to skin in the Reconstructed Human Epidermis (RhE) test method.

Executive summary:

A study was conducted to determine the skin corrosion potential of the test substance using the Reconstructed Human Epidermis (RHE) test method according to OECD Guideline 439, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated with the test substance for 60 min. The substance was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control, 5% SDS solution as positive control. After treatment with the test substance or the positive / negative control, cell viability of the tissues was evaluated by addition of MTT. The absorbance values for the negative control were within the required acceptability range (OD = 2.0). The positive control showed clear irritating effects (elative absorbance was reduced to 1.8%). Variation within the tissue replicates was acceptable. Under the study conditions, after the treatment with the test substance, the relative absorbance values were reduced to 99.4 %. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance is considered as non-irritant to skin in the Reconstructed Human Epidermis (RhE) test method (Andres, 2017).

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:

Ferbuary 15, 2017

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)

Specific details on test material used for the study:

Batch No.: 1591ZG-101; CAS No.: 162492-07-1; EINECS No.: 500-740-9; Purity: >98 %; Appearance: homogeneous, white powder

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

Specification:
Species Bos primigenius Taurus (fresh bovine corneas)

Origin:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH. 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 hour.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Neat test substance tested in three replicates:
Replicate 1: 643.3 mg;
Replicate 2: 616.8 mg;
Replicate 3: 667.6 mg;

Duration of post- treatment incubation (in vitro):

4 h at 32 ± 1 °C

Details on study design:

Preparations:
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C. On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

Method description:
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 solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, test substance or positive control solution were applied to each replicate.

Open Chamber Method:
The “open chamber-method” is used for solid substances. In order to apply the test substance, the nut was unscrewed to remove the glass disc. The test substance could be applied directly on the cornea now. The following amounts of the test substance were tested neat and applied directly on the cornea using a weighing funnel: 643.3 mg, 616.8 mg, 3 667.6 mg. The test substance was applied on the epithelium in such a manner that as much as possible of the cornea was covered with test substance. Exposure time on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded. The cMEM without phenol red was then removed from the front chamber and 1 mL sodium fluorescein solution (concentration: 5 mg/mL) was added to the front chamber. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid at 492 nm.

Irritation parameter:

in vitro irritation score

Value:

-0.39

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

According to the guideline, the test is considered valid if the positive control causes an IVIS that falls within the two standard deviations of the current historical mean value. The negative control has to show an IVIS ≤ 3.
IVIS of negative control HBSS-solution: 2.13 (criterion: ≤ 3)
IVIS of positive control 20% imidazole solution: 113.47 (criterion: 73.75 – 163.55)
Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance showed no effects on the cornea of the bovine eye. The calculated IVIS was established at -0.39.

Executive summary:

A study was conducted to determine the corneal damage potential of the test substance according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage by the substance is assessed by quantitative measurements of changes in corneal opacity and permeability. Bovine corneas were collected from slaughtered cattle which were between 12 and 60 months old. One valid experiment was performed with three replicates for negative control, positive control and the test substance. The neat test substance was brought onto the cornea of bovine eyes which had been incubated with cMEM without phenol red at 32 ± 1°C for 1 h and whose opacity had been measured. The test substance was incubated on the cornea for 4 hours at 32 ± 1°C. After removal of the test substance, opacity and permeability values were measured. HBSS-solution was used as negative control. Imidazole solution was used as positive control. The negative control showed no irritating effect on the cornea and the calculated IVIS was 2.13. The positive control induced serious eye damage on the cornea and fell within the standard deviations of the current historical mean values. The calculated IVIS was 113.47. Under the study conditions, the test substance showed no effects on the cornea of the bovine eye. The calculated IVIS was established at -0.39 (Andres 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

A study was conducted to determine the skin corrosion potential of the test substance using the Reconstructed Human Epidermis (RHE) test method according to OECD Guideline 439, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated with the test substance for 60 min. The substance was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control, 5% SDS solution as positive control. After treatment with the test substance or the positive / negative control, cell viability of the tissues was evaluated by addition of MTT. The absorbance values for the negative control were within the required acceptability range (OD = 2.0). The positive control showed clear irritating effects (elative absorbance was reduced to 1.8%). Variation within the tissue replicates was acceptable. Under the study conditions, after the treatment with the test substance, the relative absorbance values were reduced to 99.4 %. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance is considered as non-irritant to skin in the Reconstructed Human Epidermis (RhE) test method (Andres, 2017).

Eye

A study was conducted to determine the corneal damage potential of the test substance according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage by the substance is assessed by quantitative measurements of changes in corneal opacity and permeability. Bovine corneas were collected from slaughtered cattle which were between 12 and 60 months old. One valid experiment was performed with three replicates for negative control, positive control and the test substance. The neat test substance was brought onto the cornea of bovine eyes which had been incubated with cMEM without phenol red at 32 ± 1°C for 1 h and whose opacity had been measured. The test substance was incubated on the cornea for 4 hours at 32 ± 1°C. After removal of the test substance, opacity and permeability values were measured. HBSS-solution was used as negative control. Imidazole solution was used as positive control. The negative control showed no irritating effect on the cornea and the calculated IVIS was 2.13. The positive control induced serious eye damage on the cornea and fell within the standard deviations of the current historical mean values. The calculated IVIS was 113.47. Under the study conditions, the test substance showed no effects on the cornea of the bovine eye. The calculated IVIS was established at -0.39 (Andres 2017).

Justification for classification or non-classification

Based on the results of in vitro testing, the test substance does not need to be classified for skin or eye irritation according to CLP (EC 1272/2008) criteria.