Carbamic acid, [(1S)-2-[(2-aminoethyl)amino]-1-[(4-ethoxyphenyl)methyl]ethyl]-, phenylmethyl ester, dihydrochloride

Endpoint summary

Description of key information

Skin irritation (rat): irritating [report, Lewin 2004]
Eye irritation (BCOP): corrosive [report, Gmelin 2014]

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

Study period:

from February to April 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

24 April 2002

Deviations:

yes

Remarks:

- exposure time 24 hrs instead of 4 hrs; number of animals 6 instead of 3 animals

Principles of method if other than guideline:

A combined study on acute dermal toxicity and on local tolerance was used to assess skin irritation/corrosion. The study design and reporting deviates from the current OECD TG 404, however, the study is considered sufficient for assessment of this endpoint.

GLP compliance:

yes

Species:

rat

Strain:

other: Han:WIST (SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Schering AG, Germany
- Mean weight at study initiation: 287-314 g (males) or 198-205 g (females)
- Housing: 1 animal per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: >= 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23
- rel. Humidity (%): 48 - 57
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

occlusive

Preparation of test site:

not specified

Vehicle:

other: 9 mg NaCl ad 1 mL, aqua p.i.

Controls:

other: the untreated skin served as control

Amount / concentration applied:

79.2 - 125.8 mg (= 400 mg/kg bw doese group animals)
400.0-590.2 mg/animal (= 2000 mg/kg bw dose group animals)

Duration of treatment / exposure:

24 hours

Observation period:

14 days

Number of animals:

3 males and 3 females

Details on study design:

TEST SITE
- Area of exposure: 4 x 9 cm (approximately 10% of the body surface area of a rat)
- % coverage: 10%

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no removal


OBSERVATION TIME POINTS
(indicate if minutes, hours or days) 1, 24, 48, and 72h; total observation period 14 days

SCORING SYSTEM: According to Amendment of the Directive 67/548/EEC, Annex V, B.4. Acute toxicity (Skin irritation) in the version of the Directive 92/69/EEC, dated 31 Jul 1992.

Irritation parameter:

erythema score

Basis:

animal: #1-#6

Time point:

24/48/72 h

Score:

3.5

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal: #1- #6

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable

At the dose of 2000 mg/kg the animals showed primary and secondary efflorescences.

		Animal number and sex
Location (finding)	Time-point after end of exposure	27M	28M	29M	30F	31F	32F
Reddening and scrab formation	1 h	0	0	0	1	1	1
	24 h	4	4	4	1	4	4
	48 h	4	4	4	1	4	4
	72 h	4	4	4	1	4	4
Individual mean values:		4	4	4	1	4	4
Mean value of all animals							3.5
Swellings	1 h	0	0	0	0	0	0
	24 h	0	0	0	0	0	0
	48 h	0	0	0	0	0	0
	72 h	0	0	0	0	0	0
Individual mean values:		0	0	0	0	0	0
Mean value of all animals							0

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

A combined study on acute dermal toxicity and on local tolerance was conducted . The study design and reporting deviates from the current OECD TG 404, however, the study was considered sufficient for assessment of skin irritation/corrosion. After a single dermal administration of the test substance for 24 hours to male and female rats at a dose of 2000 mg/kg local irritation was described for 6/6 animals. Following grading at 24, 48 and 72 hours after patch removal a mean value of 3.5 for erythema/eschar was calculated. All animals were without local findings within 14 days.

Executive summary:

In a primary dermal irritation study similar to OECD TG 404, young adult Wistar rats, female and male (3/sex) were dermally exposed to 79.2 - 125.8 mg or 400.0-590.2 mg/ animal of Z-Triamine Dihydrochloride in physiological saline for 24 hours. Animals then were observed for 14 days. Irritation was scored by the method according to Amendment of the Directive 67/548/EEC, Annex V, B.4. Acute toxicity (Skin irritation) in the version of the Directive 92/69/EEC, dated 31 Jul 1992..

 

At the dose of 2000 mg/kg the animals showed primary and secondary efflorescences.. These irritations lasted 14 days. In this study, the test item is considered a dermal irritant.

 

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:

March 2014 to March 2015

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

26 July 2013

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- Number of animals: at least 5
- Characteristics of donor animals (e.g. age, sex, weight): not reported
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transport: 1L containers with 500ml HBSS and 1% penicillin / streptomycin solution, transport of the containers in coolers on ice
- Indication of any existing defects or lesions in ocular tissue samples: 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 defects were transferred into new containers with fresh HBSS solution supplemented with 1% penicillin / streptomycin solution and 1 % FBS and stored overnight at temperature (2-8°C). Eyes with defects were sorted out and disposed of.
- Selection and preparation of corneas: For the preparation of the cornea the sclera was incised with a scapel and cut by scissors. A 2-3 mm wide wide scleral edge was left around the cornea for further handling. The isolated corneas were placed with the epithelium side down into a prepared cup filled with MEM medium supplemented with 1% penicillin / streptomycin solution and 1% FBS. Each cornea was placed into 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 were placed for at least 1hour in the incubator at 32°C (±1°C).
Selection: After 1 h, the MEM medium was aspirated and the chambers were filled with fresh MEM medium. For each cornea the reference opacity value was measured before application. The measured values were put into an Excel sheet and the mean and the standard deviation calculated. Six corneas with values within the range of mean ± standard deviation were selected for the application and assigned to the positive control group and the test item group. Three corneas with the highest freference opacity values were selected for the negative control group.

Vehicle:

water

Controls:

other: vehicle control: distilled water; positive control: imidazole (20 % w/v)

Amount / concentration applied:

750 µl per cornea and chamber
test item: at 20% w/v
positive control (imidazol): at 20% w/v

Duration of treatment / exposure:

opacity: 4 hours (test item, positive/vehicle control)
permeability: 90 minutes (sodium fluorescein sodium solution)

Number of animals or in vitro replicates:

each treatment group (test item, positive and vehicle control): 3 corneas

Details on study design:

closed-chamber method was applied

NUMBER OF REPLICATES: triplicate

NEGATIVE CONTROL USED: distilled water

POSITIVE CONTROL USED: 20% (w/v) imidazole solution

APPLICATION DOSE AND EXPOSURE TIME: 20 % (w/v) in distilled water (suspension), 4 hours at 32°C

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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). Before each measurement the opacitomcter was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of triplicates

Value:

78.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Results after 4 hours exposure period

Treatment group	Opacity value^1		Permeability value^2(OD490)		In vitro irritation score (IVIS)^3
		mean		mean		mean
Vehicle control	9.8	8.5	0.011	0.009	9.9	8.1
	8.7		0.008		8.8
	7.0		0.008		7.2
Positive control	62.7^*	70.8	1.692	1.563	88.1	94.2
	67.0^*		1.415		88.3
	82.6^*		1.581		106.3
Z-Triamin-Dihydrochlorid	65.9^*	78.8	0.006	0.002	66.0	78.8
	73.4^*		0.001		73.4
	97.2^*		0.000		97.2

^*corrected values

¹The opacity values were calculated by applying the following formulae:

• Opacity = (I₀/I-0.9894)/0.0251
• Opacity value = opacity after application - opacity before application
• Corrected opacity value = opacity change - mean opacity change NC
• Mean opacityvalue = mean of all corrected opacity changes per group

²The permeability values were calculated by applying the following formulae:

• OD₄₉₀change = OD₄₉₀value - mean blank value OD₄₉₀
• Corrected OD₄₉₀change = OD₄₉₀change - mean OD₄₉₀change NC
• Mean OD₄₉₀= mean of all corrected OD₄₉₀changes per group

³Calculation of In Vitro Irritancy Score (IVIS):

• IVIS per cornea = corrected opacity value + (15 x corrected OD₄₉₀change)
• IVIS per group = mean of IVIS values per cornea in a group

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test substance was investigated in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437. The in vitro test method is used to identify chemicals inducing serious eye damage. Concentrations of 0 (vehicle control) and 20 % (w/v) formulated in distilled water were tested applying the closed chamber method. The positive control (20% w/v imidazole in distilled water) showed clear opacity and distinctive permeability of the corneas. Relative to the vehicle control, the test substance did not cause increase the corneal permeability but corneal opacity occurred. The calculated mean in vitro irritation score (IVIS) was 77.8 for the test substance, compared to > 55 for classification as inducing serious eye damage.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Z-Triamine Dihydrochloride (100% a.i.) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 July 2013.

 

The corneae were incubated with the test substance and controls for 4 hours. After rinsing with phenol red containing medium and once with pure MEM medium, the Opacity and permeability were determined. The test was performed in triplicates. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

The test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro score was 78.8.

 

The positive control (Imidazole) increased the opacity and permeability of the corneae (mean in vitro score 70.8) corresponding to a classification as corrosive to the eye. With the negative control (distilled water) neither an increase of opacity nor permeability of the corneae mediated by treatment could be observed (mean in vitro score 8.05).

 

Since the mean in vitro irritancy score of the test substance was >55.1, Z-Triamine Dihydrochloride is considered to be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

In a primary dermal irritation study similar to OECD TG 404, young adult Wistar rats, female and male (3/sex) were dermally exposed to 79.2 - 125.8 mg or 400.0-590.2 mg/ animal of Z-Triamine Dihydrochloride in physiological saline for 24 hours. Animals then were observed for 14 days. Irritation was scored by the method according to Amendment of the Directive 67/548/EEC, Annex V, B.4. Acute toxicity (Skin irritation) in the version of the Directive 92/69/EEC, dated 31 Jul 1992.

At the dose of 2000 mg/kg the animals showed primary and secondary efflorescences.. These irritations lasted 14 days. In this study, the test item is considered a dermal irritant.

 

 

Respiratory irritation: no data available

 

Eye irritation:

This in vitro study was performed to assess the corneal irritation and damage potential of Z-Triamine Dihydrochloride (100% a.i.) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 26 July 2013.

 

The corneae were incubated with the test substance and controls for 4 hours. After rinsing with phenol red containing medium and once with pure MEM medium, the Opacity and permeability were determined. The test was performed in triplicates. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

The test item caused an increase of the corneal opacity and permeability. The calculated mean in vitro score was 78.8.

 

The positive control (Imidazole) increased the opacity and permeability of the corneae (mean in vitro score 70.8) corresponding to a classification as corrosive to the eye. With the negative control (distilled water) neither an increase of opacity nor permeability of the corneae mediated by treatment could be observed (mean in vitro score 8.05).

 

Since the mean in vitro irritancy score of the test substance was > 55.1, Z-Triamine Dihydrochloride is considered to be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: corrosive

Justification for classification or non-classification

Skin irritation:

 

mean value for erythema/eschar: 3.5 (6 rats, female/male, 24 hours)

 

According to EU-Directive 67/548/EEC, Annex VI the test substance shall be classified as irritant. According to regulation (EC) No 1272/2008, Annex I, the test substance shall be allocated to Category 2.

 

Eye irritation:

 

BCOP, positive (mean IVIS = 77.8)

According to regulation (EC) No 1272/2008, Annex I, the test substance shall be allocated to Category 1.