2,4-dioxo-1,3-diazetidine-1,3-bis(methyl-m-phenylene) diisocyanate

Endpoint summary

Description of key information

Metalink U is neither corrosive nor irritant to the skin in tests using reconstructed human epidermis (Wingenroth 2014a+b). In vitro studies with Metalink U reveal no potential to serious eye damage (BCOP; Gmelin, 2016) or to irritant effects to the eyes (HCE; Wingenroth, 2016c). In a subacute (4-week) inhalation study in rats Metalink U has an irritant effect on the lungs (Kopf, 2016) and also the clinical symptoms observed in the acute inhalation toxicity study in rats (Kopf, 2015a) gave evidence of an irritation potential at the respiratory tract..

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:

May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2014-07-27

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test method

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm


PREDICTION MODEL / DECISION CRITERIA:
- Corrosivity potential of test materials is predicted from the cell viabilities obtained after 3 min and 60 min treatment compared to the negative control. A chemical is classified "corrosive" (sub-category 1A) if the cell viability after 3 min treatment is decreased by more than 50 %. If cell viability after 3 min exposure is ≥ 50 %, while it is below 15 % after 60 min exposure the substance is also classified as corrosive, but sub-category 1 B/1 C. If cell viability after 3 min exposure is ≥ 50 % and after 60 min exposure ≥ 15 %, the substance is classified as non-corrosive.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (plus 50 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): 0.9% NaCl

Duration of treatment / exposure:

3 and 60 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

97.23

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

100.72

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Table 1: Tabular summary of the results

 

Sample No.	Test item	Time (min)	OD mean *	Std Dev	% Viability
1 - 3	Negative control NaCl 0.9 %	60	1.93	0.20	100.00
4 - 6	Metalink U	60	1.95	0.01	100.72
13 - 15	Negative control NaCl 0.9 %	3	2.02	0.15	100.00
16 - 18	Metalink U	3	1.96	0.08	97.23

 

* 6 values

Interpretation of results:

other: no corrosive property

Executive summary:

A study for predicting a non-specific, corrosive potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 431. 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.: 97.23 % viability; 60 min.: 100.72 % viability) showed, that Metalink U has no corrosive property under the conditions of the assay used.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2014

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2014-07-27

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test method

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA:

- The mean optical density (OD) values obtained with the test item were used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- according to UN GHS (Category 2) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 0.9% NaCl in water

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

Duration of treatment / exposure:

20 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

20 min

Value:

96.72

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- DEMONSTRATION OF TECHNICAL PROFICIENCY:

Reliability of the test was previously confirmed by interlaboratory validation

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

Table 1: Tabular summary of the results

Sample No.	Test item	OD mean *	Std Dev	% Viability
1 - 3	Negative control NaCl 0.9 %	2.10	0.09	100.00
4 - 6	Positive control SDS 5 %	0.03	0.00	1.24
7 - 9	Metalink U	2.03	0.05	96.72

 

* 6 values

Interpretation of results:

other: no irritant property

Executive summary:

A study for predicting a skin irritation potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 439. After an exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the mean value of cell viability was measured to be 96.72 % in the MTT (Methylthiazoletetrazolium) conversion assay. Thus, Metalink U is considered to have no skin irritation category as defined in the UN GHS.

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:

Feb 2016

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

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2016-06-09

Species:

other: isolated cornea from eyes of slaughtered cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- Extraction: Staff of the slaughterhouse
- Transport: 1L containers with 500 mL HSS and 1 % penicillin/streptomycin solution; transport of the containers in coolers on ice

Vehicle:

other: corn oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 μL per cornea

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

- PREPARATION OF CORNEAS: Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin I streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded. On the next day (day of experiment) the containers with the eyes were transferred in an incubator at 32 °C (± 1 °C) for about 2 hours. For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 %penicillin I streptomycin solution and 1 % FBS. Each cornea was placed in a cornea holder with the endothelial side on the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour into the incubator at 32 °C (± 1 °C).
- SELECTION OF CORNEAS FOR APPLICATION: Following 1 hour in the incubator, the MEM medium was aspirated and the chambers were refilled with fresh MEM medium. For each cornea the reference opacity value was measured then. The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean ± standard deviation were selected for the actual test and assigned to the test groups. The numbers of the corneas selected were documented in an appropriate protocol. The holders were labeled with a new serial number for the further testing.
- APPLICATION OF THE TEST MATERIAL AND INCUBATION: Immediately before application, the medium was aspirated from the anterior chamber. 750 µL of the test material formulations were applied to the corneas, each through the holes of the anterior chamber. The holes of both chambers were sealed with adhesive tape and the holders were kept with the front side up, so that the formulations covered the cornea sufficiently. The holders were transferred into the incubator at 32 °C (± 1 °C) for the exposure time of 4 hours. After the exposure, the formulations were aspirated from the anterior chamber. The corneas treated with the vehicle control, the negative and the positive control were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. The corneas treated with the test item were rinsed at first with com oil and then also at least 3 times with phenol red containing MEM. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. Before measuring opacity, fresh MEM medium was filled in the chambers.
- DETERMINATION OF OPACITY: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort 3.4 SP6 software from Testo AG, Lenzkirch). The validation of the opacitometer was carried out under the study number T8082017. Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- DETERMINATION OF PERMEABILITY: The medium in anterior chamber of each holder was replaced by 1 ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 oc (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 )lL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluorescein solution was prepared and also filled into the 96-well plate, in triplicates. The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA- Reader (Bio-Tek EL 808, Software Gen5).
- CALCULATION AND EVALUATION OF IN VITRO IRRITANCY SCORE (IVIS): All parameters and the IVIS values were calculated by using Microsoft Excel. The validation of the Excel file was carried out under the study number T0082019.
The opacity values were calculated by applying the following formulae:
1) Opacity= (Io/I-0.9894)/0.0251
2) Opacity change = opacity after application - opacity before application
3) Corrected opacity change= opacity change- mean opacity change NC
4) Mean opacity = mean of all corrected opacity changes per group
The permeability values were calculated by applying the following formulae:
1) OD49o change OD490 value - mean blank value OD490
2) Corrected OD490 change= OD490 change- mean OD490 change NC
3) Mean OD490 =mean of all corrected OD490 changes per group
Calculation of In Vitro Irritancy Score (IVIS):
1) IVIS per cornea= corrected opacity change+ (15 x corrected OD490 change)
2) IVIS per group mean of IVIS values per cornea in a group

Io = single value of the measurement of empty holder with medium but without cornea, measured l-2days before the experiment;
I = individual value of each opacity measurement before and after application 0.9894/0.0251 is a constant, which is required for calculation.

- DECISION CRITERIA: The IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
IVIS <= 3 (No category), IVIS > 3 - <= 55 (No prediction can be made / No Category 1) or IVIS > 55 (Category 1)

Irritation parameter:

other: in vitro irritancy score (IVIS)

Run / experiment:

4 hrs

Value:

10.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Tabular in vitro irritancy scores (IVIS)

	Cornea No.	Opacity per cornea	Permeability per cornea	IVIS per cornea	IVIS mean	SD
Negative control (0.9 % NaCl)	1	1.8	0.008	2.0
	2	- 2.1	0.007	- 2.0	- 0.3	2.0
	3	- 0.9	0.008	- 0.8
Positive control (20 % Imidazol)	4	90.0	0.928	106.5
	5	377.3	1.331	391.2	198.1	167.4
	6	76.5	0.940	96.4
Vehicle control (corn oil)	7	1.8	0.003	1.9
	8	10.0	0.003	10.9	8.2	5.5
	9	11.9	0.002	11.9
Test item (20 % Metalink U)	10	16.8	0.002	16.8
	11	4.9	0.001	4.9	10.5	6.0
	12	9.8	- 0.001	9.7

No potential for serious eye damage was concluded from the study, as the IVIS was below 55 for the test item.

Interpretation of results:

other: no severe eye damage

Executive summary:

Metalink U was investigated in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437. The epithelial surface of the corneas was exposed to 750 µL of the test substance formulated in corn oil. Measurement of corneal opacity and permeability after a 4 hours exposure followed by a post-treatment incubation of 90 minutes revealed an in vitro irritation score (IVIS) of 10.5, well below the threshold for classification of serious eye damage (IVIS <=55). The positive (20 % imidazol) and negative (saline solution) controls confirmed the validity of the test. Thus, under the conditions of this test Metalink U was characterized by having no potential to seriously damage the eye.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

The HCE model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for severe eye damage/eye irritancy (e.g. Cotovio et. al., Tox. in Vitro, 24, 2010, 523-537), and is routinely used by cosmetic and pharmaceutical companies. It has been prevalidated (van Goethem et. al., Tox in Vitro 20, 2006, 1-17; Alépée et al., Tox in Vitro 27, 2013, 1476-1488) and has entered formal ECVAM validation in 2010. Although a high reproducibility was attested within the validation process, a further need for optimization was identified. This is currently being addressed outside the validation study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 221305
- Purity: > 98.0 %
- Expiration date of the lot/batch: 2014-07-27

Species:

other: human corneal epithelium (HCE)

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 from the cell line HCE 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:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg per insert

Duration of treatment / exposure:

60 minutes

Duration of post- treatment incubation (in vitro):

16 hours

Number of animals or in vitro replicates:

3

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. 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. A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50 %.

Irritation parameter:

other: % cell viability

Run / experiment:

60 min

Value:

93.39

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Tabular summary of the results

Sample No.	Test item	OD mean *	Std Dev	% Viability
1 - 3	Negative control PBS	1.22	0.07	100.00
4 - 6	Positive control SDS 0.3 %	0.25	0.13	20.32
7 - 9	Metalink U	1.14	0.11	93.39

 

* 6 values

 

Interpretation of results:

other: non-irritant to the eye

Executive summary:

An in vitro study for assessing ocular irritation was conducted in a human corneal epithelial (HCE) cell model. This model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for serious eye damage/eye irritancy (e.g. Cotovio et al., Toxicol In Vitro 24: 523-537, 2010), and is routinely used by cosmetic and pharmaceutical companies. Undiluted test item was applied topically to the reconstructed HCE tissue (30 mg per insert plus 30 µl PBS to moisten and ensure good contact to the tissue). After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 93.93 % as measured by a MTT conversion assay. As the cut-off for a non-irritant to the eye is 50 % (a test substance is predicted to be an ocular irritant if the relative tissue viability (%) exposed to the test substance is ≤ 50), Metalink U was considered to be non-irritant to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

A study for predicting a non-specific, corrosive potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 431 (Wingenroth, 2014a). 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.: 97.23 % viability; 60 min.: 100.72 % viability) showed, that Metalink U has no corrosive property under the conditions of the assay used.

A study for predicting a skin irritation potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 439 (Wingenroth, 2014b). After an exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the mean value of cell viability was measured to be 96.72 % in the MTT (Methylthiazoletetrazolium) conversion assay. Thus, Metalink U is considered to have no skin irritation category as defined in the UN GHS.

Metalink U was investigated in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437 (Gmelin, 2016). The epithelial surface of the corneas was exposed to 750 µL of the test substance formulated in corn oil. Measurement of corneal opacity and permeability after a 4 hours exposure followed by a post-treatment incubation of 90 minutes revealed an in vitro irritation score (IVIS) of 10.5, well below the threshold for classification of serious eye damage (IVIS <=55). The positive (20 % imidazol) and negative (saline solution) controls confirmed the validity of the test. Thus, under the conditions of this test Metalink U was characterized by having no potential to seriously damage the eye.

An in vitro study for assessing ocular irritation was conducted in a human corneal epithelial (HCE) cell model (Wingenroth, 2014c). This model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for serious eye damage/eye irritancy (e.g. Cotovio et al., Toxicol In Vitro 24: 523-537, 2010), and is routinely used by cosmetic and pharmaceutical companies. Undiluted test item was applied topically to the reconstructed HCE tissue (30 mg per insert plus 30 µl PBS to moisten and ensure good contact to the tissue). After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 93.93 % as measured by a MTT conversion assay. As the cut-off for a non-irritant to the eye is 50 % (a test substance is predicted to be an ocular irritant if the relative tissue viability (%) exposed to the test substance is ≤ 50), Metalink U was considered to be non-irritant to the eye.

Justification for classification or non-classification

Skin and eye irritation

Based on the study results of in vitro skin and eye irritation/corrosion tests a classification according to Regulation (EC) No. 1272/2008 (CLP) is not required.

Respiratory irritation

Based on the study results of an acute inhalation study in rats (clinical symptoms caused by irritation) as well as a subacute (4-week) inhalation study in rats (lung effects caused by irritation) a classification according to Regulation (EC) No.1272/2008 (CLP) with STOT Single Exp. 3 ( H335: May cause respiratory irritation) is recommended.