9H-Carbazole-9-propanoic acid, 3-[[(4-fluorophenyl)sulfonyl]amino]-1,2,3,4-tetrahydro-, compd. with 1-butanamine (1:1), (3R)-

Endpoint summary

Description of key information

The assessment of the acute local tolerance of the butyl ammonium salt of an Active Pharmaceutical Ingredient (API) has been done in a weight of evidence approach using the in vivo study results of the API supplemented with additional in vitro assays with the butyl ammonium salt. The skin irritation potential of the butyl ammonium salt corresponds to that of the API. Both substances caused no irritation. With regard to the eye the butylammonium salt seems to be more harmful. Although reversible effects on the cornea and iris were also documented for 2/3 animals in the study with the API, the butylammonium salt

shows the potential to seriously damage the eye due to the result of a BCOP test. The HET-CAM assay was negative with the regard to the descision criteria but documented a moderate irritation. This corresponds well to the assumption that the adverse effect on the eye for the butylammonium salt is more marked than the effect for the API because no effects on the conjunctival tissue were observed in the in vivo study of the API.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

December 2015

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)

GLP compliance:

yes (incl. QA statement)

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 or Category 1) 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:

three

Irritation / corrosion parameter:

% tissue viability

Value:

105.11

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	StdDev	% Viability
1-3	Negative control NaCl 0.9 %	2.23*	0.12	100.00
4-6	Positive control SDS 5 %	0.03*	0.01	1.38
10, 12	Butylammonium salt (BA-Salz)	2.34#	0.04	105.11

* 6 values

# 4 values the OD values of sample 11 were not considered for the calculation of the cell viability, due to possible damage of the epidermis.

Interpretation of results:

other: negative

Executive summary:

A study was performed for the assessment of the skin irritancy of the test item with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439 and EU Test Method B.46. The mean value of cell viability was recorded to be 105 %. The test item was thus shown to be not irritating to reconstructed human skin in vitro.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-Across from experimental result for a structural analogue, OECD guideline followed 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)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sex: male
- Source: LPT Laboratory of Pharmacology and Toxicology KG, 24601 Löhndorf, Germany
- Age at study initiation: approx. 6 months
- Weight at study initiation: 2.0 - 2.4 kg
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 20 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: pulverized solids were moistened with water

Controls:

other: the surrounding untreated skin served as control

Amount / concentration applied:

500 mg

Duration of treatment / exposure:

4 hours

Observation period:

1, 24, 48 and 72 hours after patch removal

Number of animals:

3

Details on study design:

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

REMOVAL OF TEST SUBSTANCE
- Washing: no residual test item had to be removed
- Time after start of exposure: 4 hours

SCORING SYSTEM: according to DRAIZE

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24, 48, 72 hours

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24, 48, 72 hours

Score:

0

Max. score:

4

Other effects:

No systemic intolerance reactions were observed.

Interpretation of results:

not irritating

Remarks:

Migrated information

Executive summary:

In a dermal irritation study according to OECD TG 404 Bay U 3405 HS (actice pharmaceutical ingredient) was applied under semiocclusive conditions for 4 hours to the shaved skin of 3 rabbits. Skin irritation was assessed after 1, 24, 48 and 72 hours using the Draize scale. The mean irritation index for erythema and edema was determined with 0.0, thus, the test material was not irritating to the skin of rabbits. No systemic intolerance reactions were observed.

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:

weight of evidence

Study period:

January 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)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: Analytical determination of stability and homogeneity of the test item in the vehicle was not performed specifically for this study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The usage of physiologic saline solution for formulation of the test item is plausible based on
the current knowledge of the sponsor. No objections were seen particularly in regard to stability.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was suspended by using a mortar shortly prior to application in physiologic
saline solution, the formulation was continuously stirred.
- Final preparation of a solid: 20% (w/v)

FORM AS APPLIED IN THE TEST (if different from that of starting material): The preparation was visually described as suspension.

Details on test animals or tissues and environmental conditions:

Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s balanced salt solution (HBSS) with penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HBSS supplemented with penicillin/streptomycin solution and 1 % FBS and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 ° C (± 1 ° C) for about 2 hours before preparation of the corneas.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL per cornea and chamber
- Concentration (if solution): 20 % (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL per cornea and chamber
- Concentration (if solution): 0.9% NaCl (w/v)

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

no further incubation required

Number of animals or in vitro replicates:

3 cornea

Details on study design:

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 (closed chamber method). 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 and the corneas were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. 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 and the holes were sealed with tape.

Positive control: 20 % Imidazole in physiologic saline (w/v; 750 µL)

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal 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). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability:The medium in anterior chamber of each holder was replaced by 1ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 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 μL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluoresceinsolution 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).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:decision criteria as indicated in the OECD TG 437

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

82.7

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Individual values of opacity, permeability and IVIS

Cornea No.	Opacity per cornea	Permeability per cornea	IVIS per cornea	IVIS per group
				mean		SD
Vehicle control                    1	-0.6	0.006	-0.5
0.9 % NaCl                              2	-0.1	0.006	0.0	-0.9		1.2
3	-2.4	0.008	-2.3
Positive Control                     4	77.3	1.004	92.3
20 % Imidazole                      5	60.5	1.122	77.4	87.1		8.4
6	75.1	1.091	91.5
Test item                                7	48.2	2.326	83.1
20% Butylammonium           8	56.4	2.226	89.8	82.7		7.3
salt                                           9	40.6	2.299	75.1

 

Interpretation of results:

other: positive

Executive summary:

The test item (20 % (w/v) in physiologic saline) was tested in the BCOP test according to OECD TG 437. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour exposure time. Based on these data, in comparison to the vehicle control, the In Vitro Irritancy Score (IVIS) value was calculated to be 82.7 which is above the IVIS cut-off threshold of 55 and thus identifying the test item as inducing serious eye damage. The results of the positive (20 % imidazole solution) and vehicle (physiologic saline solution) controls confirmed the validity of the test system.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: Appendix B3 of "ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products", NIH Publication No. 10-7553, September 2010.

Principles of method if other than guideline:

- Principle of test: The Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) is a test method which implies the use of a complete tissue constituted of blood vessels and proteins that is capable of responding to chemical injury with an inflammatory process similar to the one occuring in the conjunctival tissue of the eye.

- Short description of test conditions: The test substance is applied directly to the chorioallantoic membrane (CAM) of fertilized chicken eggs

- Parameters analysed / observed: acute effects on haemorrhage, lysis of blood vessels and coagulation

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

SOURCE OF FERTILIZED CHICKEN EGGS
- Source: Brinkschulte Josef GmbH & Co.KG, 48308 Senden
- Number of eggs: 4
- Characteristics of donor animals fertile Lohmann Brown hens
- Treatment conditions of eggs prior initiating testing: day 1-7: an incubator with an automatic rotating device (e.g. Ehret GmbH), optimum temperature : 37.5 °C, relative humidity 63%. day 8: with the large end
upward and not rotated for ensuring accessibility to the Chorioallantoic membrane (CAM) region
- Time interval prior to initiating testing: 8 days
- indication of any existing defects or lesions in eggs: After 7 days of incubation, all eggs were candled in order to discard those that were defect and to mark the air bubble.

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): 300 μl/egg which corresponded to an amount from an average of 45 mg

Duration of treatment / exposure:

300 sec

Duration of post- treatment incubation (in vitro):

not applicable

Number of animals or in vitro replicates:

4 eggs

Details on study design:

At day 8 of incubation the sections marked for the air bubble were sawed out of the shell. The inner membrane was moistened with NaCl 0.9 % and carefully removed with forceps. Only eggs with normally developed embryos and blood vessel systems were used for testing. Undilutet test item was applied directly onto the Chorioallantoic membrane (CAM) of each egg in a volume of 300 µL undiluted test item. 4 eggs each were used for the test item, negative and positive controls.

Observations of effects to the blood vessels, albumen or embryo over a period of 300 seconds after substance application are determined for each single egg. The time to the appearance of each of the observations mentioned above has been monitored and recorded. If no effect appeared during the observation period of 300 seconds (observation = 0) the result was assigned as negative for the related endpoint, and the factor set to 0 for this endpoint when calculating the Irritation Score (IS).

Scoring criteria for the acute effects and calculation of Irritancy Score (IS):

0 = no effect
1 = vasodilatation, slight haemorrhage (H)
2 = vessel lysis, strong haemorrhage (L)
3 = blood-coagulation, albumen-coagulation (C)

IS = 5 x (301-sec H)/300 + 7 x (301- sec L)/300 + 9 x (301- sec C)/ 300

H= observed start in seconds of haemorrhage reactions; L= observed start in seconds of vessel lysis, strong haemorrhage; C= observed start in seconds of blood - coagulation, albumen - coagulation

Data Interpretation:

Irritation Score (IS)

0-0.9 -> Non irritant
1-4.9 -> Slight irritant
5-8.9 -> Moderate irritant
9-21 -> Strong irritant

A test substance is considered to cause severe irritation when the IC value is greater than nine.

Irritation parameter:

other: irritation score (IS)

Run / experiment:

mean

Value:

6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A test substance is considered to cause severe irritation when the IC value is >= 9.

Table 1: Summary of results, test item

Egg	Effect	Effect detected after [sec]	Irritation Score (IS)
1	1	51
	2	160
	3	> 300	7
2	1	110
	2	213
	3	> 300	5
3	1	45
	2	142
	3	> 300	8
4	1	108
	2	227
	3	300	5

effect: 1 = vasodilatation, slight haemorrhage 2 = vessel lysis, strong haemorrhage 3 = blood-coagulation, albumen-coagulation

Interpretation of results:

other: negative

Executive summary:

The test item was tested in the in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method. This is a method that makes use of the chorioallantoic mambrane (CAM) of fertilized chicken eggs. For determination of acut effects on haemorrhage, lysis of blood vessels and coagulation the undiluted test item was directly applied onto the CAM. for 5 minutes. The Irritation Score (IS) value was calculated to be 6 for the test item which was interpreted as being a moderate irritant. The IS value of 6 does not reach or exceed the cut-off threshold of 9 and thus identifying the test item as not inducing serious eye damage.The results of the positive ( SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across, GLP 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

Species:

rabbit

Strain:

Himalayan

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Sex: male
- Source: LPT Laboratory of Pharmacology and Toxicology KG, 24601 Löhndorf, Germany
- Age at study initiation: approx. 4.5 months
- Weight at study initiation: 2.2 - 2.4 kg
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 20 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

unchanged (no vehicle)

Controls:

other: the left eye, which remained untreated, served as control

Amount / concentration applied:

100 mg

Duration of treatment / exposure:

eye was not rinsed after instillation of the test item

Observation period (in vivo):

1, 24, 48 and 72 hours and 4 to 21 days after instillation

Number of animals or in vitro replicates:

3

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: #2 and #3

Time point:

other: 24, 48, 72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 6 days (animal 3) or 8 days (animal 2)

Remarks on result:

other: corneal staining became obvious 24 h after treatment in animal 2 (whole surface) and animal 3 (3/4 of surface) and 7 days after treatment in animal 2 (1/4 of the surface)

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 24, 48, 72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal: #2 and #3

Time point:

other: 24, 48, 72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 5 days (animal 3) or 8 days (animal 2)

Irritation parameter:

conjunctivae score

Remarks:

redness and chemosis

Basis:

mean

Time point:

other: 24, 48, 72 h

Score:

0

Other effects:

No systemic intolerance reactions were observed.

Interpretation of results:

irritating

Remarks:

Migrated information

Executive summary:

In an eye irritation study according to OECD TG 405 the test substance was instilled into the conjunctival sac of one eye of 3 male rabbits each. Eye irritation was assessed 1, 24, 48 and 72 hours after instillation using the Draize scale. In this study irritation was seen as corneal opacity (up to grade 1 of max. 4 in 2 of 3 animals) and effects on iris (up to grade 1 of max. 2 in 2 of 3 animals). The effects appeared to be reversible within <= 6 days after treatment. No systemic intolerance reactions were observed.

According to classification criteria Bay U 3405 is irritating to rabbit's eyes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation:

BA-Salz is the butylammonium salt of the an Active Pharmaceutical Ingredient (API) for which the acute local irritation potential was examined in a dermal irritation study according to OECD TG 404. The API was applied under semiocclusive conditions for 4 hours to the shaved skin of 3 rabbits. Skin irritation was assessed after 1, 24, 48 and 72 hours using the Draize scale. The mean irritation index for erythema and edema was determined with 0.0, thus, the test material was not irritating to the skin of rabbits. No systemic intolerance reactions were observed.

Due to the butylammonium moity of the salt of the API an additional in vitro test was performed with the salt to exclude the possibility that the local tolerance of the salt differs from the API, which is non-irritant to the skin.

A study was performed for the assessment of the skin irritancy of the butyl ammonium salt of the API with reconstructed human epidermis (RhE). The experiment was carried out using the commercially available test method epiCS®. The study was conducted in accordance with OECD TG 439. The mean value of cell viability was recorded to be 105 %. The test item was thus shown to be not irritating to reconstructed human skin in vitro.

Eye irritation:

BA-Salz is the butylammonium salt of the an Active Pharmaceutical Ingredient (API) for which the acute local irritation potential was examined in an ocular irritation study according to OECD TG 405. The API was instilled into the conjunctival sac of one eye of 3 male rabbits each. Eye irritation was assessed 1, 24, 48 and 72 hours after instillation using the Draize scale. In this study irritation was seen as corneal opacity (up to grade 1 of max. 4 in 2 of 3 animals) and effects on iris (up to grade 1 of max. 2 in 2 of 3 animals). The effects appeared to be reversible within <= 6 days after treatment. No systemic intolerance reactions were observed.

Due to the butylammonium moity of the salt of the API additional in vitro tests were performed with the salt of the API to exclude the possibility that the local tolerance of the salt differs from the API, which is irritant to the eyes.

The test item (20 % (w/v) in physiologic saline) was tested in the BCOP test according to OECD TG 437. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour exposure time. Based on these data, in comparison to the negative control, the In Vitro Irritancy Score (IVIS) value was calculated to be 82.7 which is above the IVIS cut-off threshold of 55 and thus identifying the test item as inducing serious eye damage. The results of the positive (20 % imidazole solution) and negative (physiologic saline solution) controls confirmed the validity of the test system.

The test item was tested in the in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method. This is a method that makes use of the chorioallantoic mambrane (CAM) of fertilized chicken eggs. For determination of acut effects on haemorrhage, lysis of blood vessels and coagulation the undiluted test item was directly applied onto the CAM. for 5 minutes. The Irritation Score (IS) value was calculated to be 6 for the test item which was interpreted as being a moderate irritant. The IS value of 6 does not reach or exceed the cut-off threshold of 9 and thus identifying the test item as not inducing serious eye damage.The results of the positive ( SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system.

Justification for classification or non-classification

Based on the entire data for the Active Pharmaceutical Ingredient ( API) and its butyl ammonium salt no classification for skin corrosion/irritation according to Regulation (EC) No. 1272/2008 (CLP) is required for the butyl ammonium salt.

Based on the entire data for the Active Pharmaceutical Ingredient ( API) and its butyl ammonium salt a classification with Eye Damage, Cat. 1 (H318: causes serious eye damage) according to Regulation (EC) No. 1272/2008 (CLP) is required for the butyl ammonium salt.