Methylidynetri-p-phenylene triisocyanate

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: LPT Laboratory of Pharmacology and Toxicology KG, 24601 Löhndorf/Post Wankendorf, Germany
- Age at study initiation: appr. 4.5 months
- sex: males

Type of coverage:

semiocclusive

Preparation of test site:

other: shaved intact

Vehicle:

unchanged (no vehicle)

Controls:

other: surrounding untreated skin served as control

Amount / concentration applied:

Amount: 0.5 ml

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48, 72 hours,  and 4 to 13 days after patch removal

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: approx. 6 cm²
- Type of wrap if used: non irritating tape

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 4 hours

SCORING SYSTEM: Draize

Irritation parameter:

other: light brown discolouration

Basis:

other: all 3 animals

Time point:

other: from patch removal up to 8 days

Remarks on result:

other: Due to light brown discolouration of the application area no evaluation of erythema and oedema formation was possible.

Irritation parameter:

other: induration

Basis:

animal #1

Time point:

other: 1 day up to 5 days

Remarks on result:

other: see above

Irritation parameter:

other: induration

Basis:

animal #2

Time point:

other: 3 up to 5 days

Remarks on result:

other: see above

Irritation parameter:

other: induration

Basis:

animal #3

Time point:

other: up to 5 days

Remarks on result:

other: see above

Irritation parameter:

other: laceration

Basis:

animal: #1, #3

Time point:

other: 6 to 9 days

Remarks on result:

other: see above

Irritation parameter:

other: laceration

Basis:

animal #2

Time point:

other: 5 to 8 days

Remarks on result:

other: see above

Irritation parameter:

other: peeling

Basis:

animal #1

Time point:

other: 10 to 12 days

Remarks on result:

other: see above

Irritation parameter:

other: peeling

Basis:

animal #2

Time point:

other: 9 to 10 days

Remarks on result:

other: see above

Irritation parameter:

other: peeling

Basis:

animal #3

Time point:

other: 10 and 11 days

Remarks on result:

other: see above

Irritation parameter:

other: erythema/oedema score

Basis:

animal: #1, #2, #3

Time point:

other: 9 days to 13 days after patch removal

Score:

0

Max. score:

4

Other effects:

There were no systemic intolerance reactions.

Conclusions:

Classification: not irritating

Executive summary:

Triphenylmethane-4,4',4''-triisocyanate (applied as a 27% solution in ethyl acetate) was shown to be irritating to the rabbit's skin in a study performed according to OECD TG 404. Due to light brown discolouration of the application area no evaluation of erythema and oedeme formation was possible in all 3 animals from the time point of patch removal to 8 days after patch removal. Thereafter the erythema/oedema score was 0 in all animals. On the other hand induration, laceration and peeling was observed in all animals whereas the onset of the effects differ from 24 hours to 5 days after patch removal. All effects were reversible within 13days after patch removal. No systemic intolerance reactions were observed.

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:

other: GLP guideline study

Principles of method if other than guideline:

The HCE model is currently involved in the eye irritation validation conducted by COLIPA following ECVAM guidelines. Furthermore, it is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). This model is recognized as the model of choice and scientifically relevant as documented by several publications (Cotovio et. al., Tox. in Vitro, 24, 2010, 523-537).

GLP compliance:

yes

Species:

other: Assessment of ocular irritation potential of the test substance by determination of cytotoxic effects on a human corneal epithelium (HCE) cell model (exposure 60 min./rt followed by 16 hours incubation at 37 °C, subsequently MTT).

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

The experiment was carried out on a Human Corneal Epithelial (HCE) Model, which is standardized and commercially available (SkinEthic, France). Inserts were of 0.5 cm² size. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

Vehicle:

other: ethyl acetate

Controls:

other: as negative control: Phosphate Buffered Saline (PBS, 30 µL) as vehicle control: ethyl acetate

Amount / concentration applied:

30 µl per insert

Duration of treatment / exposure:

60 min at room temperature

Observation period (in vivo):

post-exposure incubation: 16 hours (37°C, 5% CO2, maximum humidity)

Number of animals or in vitro replicates:

not applicable

Details on study design:

The irritation potential of the test item is assessed by determination of its cytotoxic effect on an reconstructed human ocular epithelia. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item.
Tests were performed in triplets. The test item was applied at a 100% concentration, i.e. 30 µl per insert, for 60 min at room temperature. After the exposure period the inserts were washed carefully with PBS. After a post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity) MTT reduction was performed. Cell viability was measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative or positive control substances or the test item.

Positive control: 1H-1,2,4-Triazole-3-thiol (30 mg, plus 30 µl PBS for moistening)

It is not advisable to test isocyanates in aqueous vehicles. Therefore, ethyl acetate had been chosen to moisture the test item before application to the cornea epithelium. Ethyl acetate is classified as an eye irritant which was confirmed by the results of this assay. The results of this assay show that the test substance Triphenylmethane-4,4’,4’’-triisocyanate also has an irritant effect, which however increases the irritant property of ethyl acetate.

Therefore the test item was detected as irritant to the cornea epithelium under the conditions of this test (s. Table 1)

Table1: Summary of results

Compound	Cell Viability [%]	Evaluation
negative control PBS	100.00	non-irritant
positive control 1H-1,2,4 -Triazole-3 -thiol	24.41	irritant
test substance	2.76	irritant

Interpretation of results:

other: irritant property in vitro

Executive summary:

An in vitro study for assessing ocular irritation of chemicals using a human 3D-corneal epithelial (HCE) cell model was conducted with Triphenylmethane-4,4',4''-triisocyanate. The cell model, routinely used by the major Cosmetic and Pharmaceutical companies, has been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). The pure test item (30 mg per insert) was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes followed by a 16 hours post-treatment incubation period the cell viability was measured as about 3 % by the MTT conversion assay. A test item is predicted to be an ocular irritant if the mean relative cell viability is ≤ 50 % after exposure. Therefore it is concluded that Triphenylmethane-4,4´,4´´-triisocyanate is an ocular irritant under the conditions of this test method.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Triphenylmethane-4,4',4''-triisocyanate obtained in a solvent-based manufacturing process and is not isolated throughout the process. A solvent-freeTriphenylmethane-4,4',4''-triisocyanate is a solid which would significantly complicate the manufacturing process. Beyond that Triphenylmethane-4,4',4''-triisocyanate is not marketed as such but as a solution to facilitate handling of the substance by downstream users. A solution of 27% Triphenylmethane-4,4',4''-triisocyanate in ethyl acetate (w/w) represents the trade product for which a base set of toxicological information was already available. On the other hand, as displayed in IUCLID section 1.4, removal of the solvent from the trade product invariably leads to the formation of higher molecular weight species, due to the inherent reactivity of the isocyanate moieties. Further, analysis of the toxicity profile of the solvent in the trade product (= ethyl acetate: non irritating to the skin and irritating to the eyes) demonstrates that the available studies with the trade product reflect the hazard of Triphenylmethane-4,4',4''-triisocyanate (= active ingredient in the trade product). To preclude corrosion/irritation for Triphenylmethane-4,4',4''-triisocyanate in concentrations greater 27% appropiate in vitro studies were performed with the solvent free solid test material.

Skin irritation:

Triphenylmethane-4,4',4''-triisocyanate (applied as a 27% solution in ethyl acetate) was shown to be irritating to the rabbit's skin in a study performed according to OECD TG 404. Due to light brown discolouration of the application area no evaluation of erythema and oedeme formation was possible in all 3 animals from the time point of patch removal to 8 days after patch removal. Thereafter the erythema/oedema score was 0 in all animals. On the other hand induration, laceration and peeling was observed in all animals whereas the onset of the effects differ from 24 hours to 5 days after patch removal. All effects were reversible within 13days after patch removal. No systemic intolerance reactions were observed.

A study for predicting non-specific, corrosive potential of Triphenylmethane-4,4',4''-triisocyanate by using reconstructed human skin (OECD TG 431) was performed. For the determination of time related cytotoxic effects the incubation periods were 3 min. and 60 min. The MTT (Methylthiazoletetrazolium) viability test results (3 min.: 108% viability; 60 min.: 24% viability compared to NaCl treated skin inserts) showed, that the test item has no corrosive property under the conditions of the assay used.

Eye irritation:

Triphenylmethane-4,4',4''-triisocyanate (applied as a 27% solution in ethyl acetate) shows irritating effects in rabbit eyes in a study performed according to OECD TG 405. The ocular reactions were restricted to 2/3 animals and were reversible within 5 days at the latest. Systemic effects were not observed.

An in vitro study for assessing ocular irritation of chemicals using a human 3D-corneal epithelial (HCE) cell model was conducted with Triphenylmethane-4,4',4''-triisocyanate. The cell model, routinely used by the major Cosmetic and Pharmaceutical companies, has been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). The pure test item (30 mg per insert) was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes followed by a 16 hours post-treatment incubation period the cell viability was measured as about 3 % by the MTT conversion assay. A test item is predicted to be an ocular irritant if the mean relative cell viability is ≤ 50 % after exposure. Therefore it is concluded that Triphenylmethane-4,4´,4´´-triisocyanate is an ocular irritant under the conditions of this test method.

Respiratory Irritation:

Triphenylmethane-4,4',4''-triisocyanateaerosol was proven to be respiratory irritating. In a pulmonary irritant potency study (TRGS 430; OECD TG 403; see IUCLID chapter 7.2.2) with aerosolizedTriphenylmethane-4,4',4''-triisocyanate, rats showed signs suggestive of respiratory tract irritation at concentrations of 1.9 mg/m³ and above. Moreover, a subacute inhalation study with aerosolized Triphenylmethane-4,4',4''-triisocyanate (OECD TG 412, see IUCLID chapter 7.5.2) revealed changes which were related to portal-of-entry, local irritant effects (larynx, airways and alveoli).

Justification for selection of skin irritation / corrosion endpoint:
The existing study in laboratory animal with the trade product (27% Triphenylmethane-4,4',4''-triisocyanate in ethyl acetate) provide sound conclusive evidence that the substance is an irritant. Corrosivity of Triphenylmethane-4,4',4''-triisocyanate concentrations greater 27% was excluded by in vitro testing.

Justification for selection of eye irritation endpoint:
The existing study in laboratory animal with the trade product (27% Triphenylmethane-4,4',4''-triisocyanate in ethyl acetate) provide sound conclusive evidence that the substance is a non-irritant. But by in vitro testing (HCE assay) the solvent free test item was irritating.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: irritating

Effect level: empty Endpoint conclusion: Adverse effect observed

Justification for classification or non-classification

Skin irritation:

No corrosive properties were demonstrated in an in vitro test for Triphenylmethane-4,4´,4´´-triisocyanate and an existing study on irritation and corrosion in laboratory animals provides sound conclusive evidence that a solution of 27% Triphenylmethane-4,4',4''-triisocyanate in ethyl acetate is an irritant:

According to EU-Directive 67/548/EEC, Annex VI Triphenylmethane-4,4',4''-triisocyanate shall be classified as irritant.

According to Regulation (EC) No 1272/2008, Annex I, Triphenylmethane-4,4',4''-triisocyanate shall be allocated to Category 2: Irritant.

Eye irritation:

While an existing study on eye irritation in laboratory animals with a solution of 27% Triphenylmethane-4,4',4''-triisocyanate in ethyl acetat demonstrates irritating effects which are not classifiable.

A validated in vitro test provides evidence that Triphenylmethane-4,4´,4´´-triisocynate is an eye irritant.

According to EU-Directive 67/548/EEC, Annex VI Triphenylmethane-4,4',4''-triisocyanate shall be classified as irritating to the eyes.

According to Regulation (EC) No 1272/2008, Annex I, Triphenylmethane-4,4',4''-triisocyanate shall be allocated to Category 2: (irritating to eyes).

Respiratory irritation:

The inhalation studies (see chapter IUCLID Chapter 7.2.2 and 7.5.2) indicate that aerosolized Triphenylmethane-4,4',4''-triisocyanate (dust) causes respiratory tract irritation.

According to EU-Directive 67/548/EEC, Annex VI Triphenylmethane-4,4',4''-triisocyanate shall be classified as irritating to respiratory system

According to Regulation (EC) No 1272/2008, Annex I, Triphenylmethane-4,4',4''-triisocyanate shall be allocated to STOT SE Category 3 for respiratory irritation.