4,4'-isopropylidenediphenyl dicyanate

Endpoint summary

Description of key information

Skin corrosion in-vitro

An in vitro skin corrosivity test was performed in a reconstructed human epidermis model, according to testing methods OECD 431 and EC B.40 bis. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Following exposure with the test item, the mean cell viability was 82.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKIN model test, the results indicate that the test item is non corrosive to the skin.

Skin irritation in-vitro

An in vitro skin irritation test was performed in a reconstructed human epidermis model, according to testing methods OECD 439 and EC B.46. The irritation of the test item was evaluated according to the OECD No. 439 guideline.

Following exposure with the test item, the mean cell viability was 99.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKIN model test, the results indicate that the test item is non-irritant to skin.

Eye irritation in-vitro

An in vitro eye irritation test was performed in a in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (26 July 2013).

No corneal swelling was observed during the four-hour observation period on test item treated eyes. Slight corneal opacity change (severity 0.5 or 1) was observed in all three eyes. Slight fluorescein retention change (severity 0.5 or 1) was noted on all three eyes. However, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. This fact might indicate morphological changes in an in vivo system (although during in vivo conditions the eyelids will probably clear the surface, but abrasion may occur). Based on these in vitro eye irritation assays in isolated chicken eyes, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

Eye irritation in-vivo

An acute eye irritation study was performed in New Zealand White rabbits, according to testing methods OECD 405 and EC B.5. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 405, 2012). Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification. The test item was placed into the conjunctival sac of the left eye of each animal. A single amount of 0.1 g of the test item was administered as a single dose. The eyes were examined 1, 24, 48, 72 hours and 7 days after application.

The test item caused conjunctival and corneal effects at one hour after application which were fully reversible within 7 days. The mean scores of irritation did not reach the critical level for classification. According to Regulation (EC) No 1272/2008, the substance does not require classification as an eye irritant.

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:

February - April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test

Deviations:

no

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test Substance Name: LZ 514
- Source: Lonza Ltd, Visp/Switzerland
- Lot/batch No. of test material: 5011
- Expiration date of the lot/batch: 2nd December 2017
- Manufacture date: 3rd December 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%), protected from light and humidity.
- Stability under test conditions: stable
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ]: The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS); conducted by the supplier.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: plates with the treated epidermis units were incubated for 4 hours at room temperature (23.6-26.5°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: rinsed thoroughly with approximately 25 mL PBS (Phosphate Buffered Saline)
- Observable damage in the tissue due to washing: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 mg test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

Duration of treatment / exposure:

4 hours incubation

Duration of post-treatment incubation (if applicable):

3 hours with MTT solution

Number of replicates:

In this assay, two replicates per test item were used. Two negative controls and two positive controls were also run in the assay. As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

two experiments

Value:

82.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no interaction between test item and MTT
- Colour interference with MTT: no

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

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with the test item, the mean cell viability was 82.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

Executive summary:

An in vitro skin corrosivity test was performed in a reconstructed human epidermis model, according to testing methods OECDE 431 and EC B.40 bis. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Following exposure with the test item, the mean cell viability was 82.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKIN model test, the results indicate that the test item is non corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - April 2016

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)

Deviations:

no

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test Substance Name: LZ 514
- Source: Lonza Ltd, Visp/Switzerland
- Lot/batch No. of test material: 5011
- Expiration date of the lot/batch: 2nd December 2017
- Manufacture date: 3rd December 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%), protected from light and humidity.
- Stability under test conditions: stable
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ]: The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS); conducted by the supplier.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: plates with the treated epidermis units were incubated for the exposure time of 15 min. at room temperature (24.1-26.1°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: rinsed thoroughly with approximately 25 mL PBS (Phosphate Buffered Saline)
- Observable damage in the tissue due to washing: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

Duration of treatment / exposure:

15 minutes incubation

Duration of post-treatment incubation (if applicable):

After rinsing, epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C.

Number of replicates:

In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

two experiments

Value:

99.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no interaction between test item and MTT
- Colour interference with MTT: no

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

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with the test item, the mean cell viability was 99.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin.

Executive summary:

An in vitro skin irritation test was performed in a reconstructed human epidermis model, according to testing methods OECDE 439 and EC B.46. The irritation of the test item was evaluated according to the OECD No. 439 guideline.

Following exposure with the test item, the mean cell viability was 99.8% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKIN model test, the results indicate that the test item is non-irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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:

- Test Substance Name: LZ 514
- Source: Lonza Ltd, Visp/Switzerland
- Lot/batch No. of test material: 5011
- Expiration date of the lot/batch: 2nd December 2017
- Manufacture date: 3rd December 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%), protected from light and humidity.
- Stability under test conditions: stable
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Species:

chicken

Strain:

other: ROSS 308

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): 30 mg test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 mg Imidazole

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL physiological saline

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

none

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Tissue: chicken eyes from chicken heads; chicken heads collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old)
- Source: TARAVIS KFT (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)
- Preparation:After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

ENVIRONMENTAL CONDITIONS
If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

NUMBER OF REPLICATES
In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

NEGATIVE CONTROL USED
Physiological saline (Salsol solution, 0.9% (w/v) NaCl)

POSITIVE CONTROL USED
Imidazole

APPLICATION DOSE AND EXPOSURE TIME
30 mg test item applied for 10 seconds

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes after the post-treatment rinse

REMOVAL OF TEST SUBSTANCE
Volume and washing procedure after exposure period: rinsed thoroughly with 20 mL physiological saline solution at ambient temperature

METHODS FOR MEASURED ENDPOINTS:
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Irritation parameter:

cornea opacity score

Run / experiment:

three test item treated eyes, three positive control treated eyes and one negative control treated eye examined

Value:

0.5 - 1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined

Value:

0.5 - 1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. This fact might indicate morphological changes in an in vivo system (although during in vivo conditions the eyelids will probably clear the surface, but abrasion may occur).

Irritant / corrosive response data:

no irritation observed

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these in vitro eye irritation assay in isolated chicken eyes, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

Executive summary:

An in vitro eye irritation test was performed in a in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (26 July 2013).

No corneal swelling was observed during the four-hour observation period on test item treated eyes. Slight corneal opacity change (severity 0.5 or 1) was observed in all three eyes. Slight fluorescein retention change (severity 0.5 or 1) was noted on all three eyes. However, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. This fact might indicate morphological changes in an in vivo system (although during in vivo conditions the eyelids will probably clear the surface, but abrasion may occur). Based on these in vitro eye irritation assays in isolated chicken eyes, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - March 2016

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Test Substance Name: LZ 514
- Source: Lonza Ltd, Visp/Switzerland
- Lot/batch No. of test material: 5011
- Expiration date of the lot/batch: 2nd December 2017
- Manufacture date: 3rd December 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%), protected from light and humidity.
- Stability under test conditions: stable
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

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: 12 weeks old
- Weight at study initiation: 2955 g – 3372 g; before euthanasia 3152 g – 3504 g
- Housing: individual housing
- Diet (e.g. ad libitum): UNI diet for rabbits produced by Cargill Takarmány Zrt., H-5300 Karcag, Madarasi út 0399, Hungary
- Water (e.g. ad libitum): municipal tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 – 24.0°C
- Humidity (%): 24 – 37%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

100 mg test item

Duration of treatment / exposure:

1 hour

Observation period (in vivo):

1, 24, 48 and 72 hours and 7 days after application

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

3 rabbits

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): none

SCORING SYSTEM:
- Draize coring system

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

scores of 3 animals

Time point:

24/48/72 h

Score:

1.67

Max. score:

2

Reversibility:

fully reversible within: 7 d

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

scores of 3 animals

Time point:

24/48/72 h

Score:

0.44

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

scores of 3 animals

Time point:

24/48/72 h

Score:

0.11

Max. score:

1

Reversibility:

fully reversible within: 48 h

Irritation parameter:

iris score

Basis:

mean

Remarks:

scores of 3 animals

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

The test item, applied to rabbit eye mucosa, caused conjunctival and corneal effects at one hour after application which were fully reversible within 7 days.

Other effects:

No Initial Pain Reaction (IPR) or any Pain Reaction (PR) was observed during the experimental period.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item caused conjunctival and corneal effects at one hour after application which were fully reversible within 7 days. The mean scores of irritation did not reach the critical level for classification. According to Regulation (EC) No 1272/2008, the substance does not require classification as an eye irritant.

Executive summary:

An acute eye irritation study was performed in New Zealand White rabbits, according to testing methods OECD 405 and EC B.5. The irritation effects of the test item were evaluated according to the Draize method (OECD No.: 405, 2012). Rabbits were treated with analgesic and anaesthetic as per the regulatory guideline. Three animals were used to make the classification. The test item was placed into the conjunctival sac of the left eye of each animal. A single amount of 0.1 g of the test item was administered as a single dose. The eyes were examined 1, 24, 48, 72 hours and 7 days after application.

The test item caused conjunctival and corneal effects at one hour after application which were fully reversible within 7 days. The mean scores of irritation did not reach the critical level for classification. According to Regulation (EC) No 1272/2008, the substance does not require classification as an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the UN Globally Harmonised System of Classification and Labelling of Chemicals, the test item does not require classification as a skin or an eye irritant.