(1R)-2-[5-(2-halo-3-methoxyphenyl)-3-[2-halo-6-(trihalomethyl)benzyl]- 4-alkyl-2,6-dioxoheterocyclyl]-1-phenylethanaminium salicylate

Endpoint summary

Description of key information

The results from the acute dermal irritation and skin irritation test analysis were negative for skin irritation of the test material.

The results from the Bovine Corneal analysis and acute eye irritation study were negative for eye irritation of the test material.

Therefore the test material did not meet the GHS criteria to be classified as a skin or eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation / corrosion, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11th June 2018 to 7th January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Acute Dermal Irritation / Corrosion (404) published by the Ministry of Environmental Protection of Peoples Republic of China in the year 2013

GLP compliance:

yes

Species:

rabbit

Strain:

other: Japanese white

Details on test animals or test system and environmental conditions:

Source: Qingdao Kangda Biological technology Limited company
A physical check up was carried out on all animals on arrival. Animals were acclimatised for 7 days and housed individually in Room A115. All animals were weighed and marked on the hair. Clinical observations were performed daily. Animals were raised in suspended stainless steel cages.
Temperature: 17-23oC
Humidity: 40-70%
Light sequence: 12 hour light / 12 hour dark
Food: Rabbit maintenance feed (Beijing keaoxieli Feed Co. Ltd.)
Water: ad libitum

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: Corn Oil

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.5g of test item moistened completely with 0.8ml vehicle

Duration of treatment / exposure:

4 hours

Observation period:

The skin sites were examined 1, 24, 48 and 72 hours after removal of patches.

Number of animals:

3

Details on study design:

0.5g of the test item was applied uniformly to the gauze patch which was a non-irritating adhesive type. The treated patch was placed on the right side of the clipped area and then covered by medical film. All animals were wrapped using elastic bandage and medical tape to form a semiocclusive dressing which gave good contact with the skin and prevented ingestion or inhalation of the test item. The left side was considered a control. Patches were removed after 4 hours. The residual test item was gently wiped off by cotton moistened with tepid water.
The skin sites were examined 1, 24, 48 and 72 hours after removal of patches for signs of skin reaction and symptoms. Dermal reactions for erythema /eschar or oedema of test sites of each animal were scored and recorded. Other local reactions and systemic side reactions were also described and recorded. Mean scores were calculated. The test item was classified according to GHS criteria for Acute Dermal Irritation / Corrosion.

Irritation parameter:

erythema score

Basis:

mean

Time point:

72 h

Score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Time point:

48 h

Score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Time point:

24 h

Score:

0

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the above results, E Stage 3 intermediate did not cause an irritation reaction in rabbits and was classified as 'unclassified' according to GHS classification criteria for skin irritation.

Executive summary:

The study was performed to assess the acute dermal irritation/corrosion of E Stage 3 intermediate in Japanese White rabbits. The method was designed to be in accordance with the guidelines for the testing of chemicals 'Acute Dermal Irritation/Corrosion' (404) published by the Ministry of Environmental Protection of People's Republic of China in the year 2013. 

Three male rabbits were used for the study. A quantity of 0.5g of the test item was applied to the right side of back skin of each animal for an exposure period of 4 hours. The untreated skin on the left back area of the animal served as the control. Clinical signs were performed once daily throughout the study. The skin sites of each animal for signs of erythema/eschar and oedema were all recorded immediately, and 1, 24, 48 and 72 hours after patch removal. All local toxic effects on skin as well as systemic toxicity were also fully described and recorded. Dermal reactions (erythema/eschar and oedema of test sites were scored at approximately 24, 48 and 72 hours after patch removal. Mean scores of erythema/eschar or oedema at 24, 48 and 72 hours were calculated for tested skins after patch removal. Individual animal body weights were recorded on the day of dosing and on the completion of fiinal observations.

No abnormal signs or symptoms were observed in any animal throughout the course of the test. The scores of erythema/eschar and oedema for all animals at approximately 24, 48 and 72 hours were all 0 after patch removal. All animals showed expected gain in bodyweight during the study. 

Based on the above results, E Stage 3 intermediate was considered to have no irritant effects on the skin of rabbits was classified as 'unclassified' according to GHS classification for eye irritation. 

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th September 2018 to 2nd January 2019

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:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

no

Remarks:

The study has been conducted with a reputable lab for other international submission.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Justification for test system used:

The protocol meets the requirements of the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439)

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after exposure to a test article.
The test article A-1298847.0, is a solid and was not evaluated in the mesh compatibility test.
The test article, the positive control (5% Sodium Dodecyl Sulfate (SDS)), and the negative control (Calcium & Magnesium Free-Dulbecco’s Phosphate Buffered Saline (CMF-DPBS)), were treated in triplicate EpiDermTM tissues for a 60±1 minute exposure period, followed by a 42-hour post-exposure expression period.
25 μL of sterile CMF-DPBS were added to the tissue surface prior to the addition of the solid test article (which was added using a 25 mg dosing spoon). The test article was mixed on the surface of the tissues using a sterile glass rod.
The MTT assay is performed by transferring the tissues to 24-well plates containing MTT medium (1 mg/mL). After a 3-hour incubation, the blue formazan salt formed by cellular mitochondria is extracted with 2ml isopropanol per tissue and the optical density of the extracted formazan is determined with a spectrometer at 570nm. Relative cell viability is calculated for each tissue as % of the mean of the negative control tissues.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Since the test article is a solid, 25 μL of sterile CMF-DPBS were added to the tissue surface prior to the addition of the solid test article (which was added using a 25 mg dosing spoon). The test article was mixed on the surface of the tissues using a sterile glass rod.

Duration of treatment / exposure:

The test article, the positive control (5% Sodium Dodecyl Sulfate (SDS)), and the negative control (Calcium & Magnesium Free-Dulbecco’s Phosphate Buffered Saline (CMF-DPBS)), were treated in triplicate EpiDermTM tissues for a 60±1 minute exposure period, followed by a 42-hour post-exposure expression period.

Duration of post-treatment incubation (if applicable):

42-hour post-exposure expression period

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

100.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The assay was accepted when the following criteria were met: 1) the positive control (5% SDS) resulted in a mean tissue viability ≤ 20%, 2) the mean OD570 value of the negative control tissues was ≥ 0.8 and < 2.8, and 3) the standard deviations of the positive and negative control calculated from individual percent tissue viabilities of the three identically treated replicates were < 18%.

Interpretation of results:

GHS criteria not met

Conclusions:

A test article was predicted to be an irritant (GHS Category 1 or 2) when the mean relative viability of the triplicate-treated tissues is ≤50% of the mean viability of the negative control. A test article was not predicted to be a skin irritant (GHS No Category) when the mean relative viability of the triplicate tissues was >50%.
The test article had a value of 100.2% ± 11.19, therefore is classed as non-irritant.

Executive summary:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after exposure to A1298847.0. The protocol was based upon the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439)2.

 

The test article, the positive control (5% Sodium Dodecyl Sulfate (SDS)), and the negative control (Calcium & Magnesium Free-Dulbecco’s Phosphate Buffered Saline (CMF-DPBS)), were treated in triplicate EpiDermTM tissues for a 60±1 minute exposure period, followed by a 42-hour post-exposure expression period. Since A-1298847.0, is a solid, 25 μL of sterile CMF-DPBS was added to the tissue surface prior to the addition of the solid test article (which was added using a 25 mg dosing spoon). The test article was mixed on the surface of the tissues using a sterile glass rod.

 

Small amounts of residual test article were noted on the tissues treated with the test article, A-1298847.0 (tissues 2 and 3 only) after the rinsing process. The test article was attempted to be removed from the exposed EpiDerm™ tissues using cotton-tipped applicators soaked in CMF-DPBS. The test article prolonged the exposure to the tissues, which may have influenced the toxic effect; however, all of the tissue viabilities were >50%, therefore, there was no significant impact on the final prediction for the test articles.

A test article was predicted to be an irritant (GHS Category 1 or 2) when the mean relative viability of the triplicate-treated tissues is ≤50% of the mean viability of the negative control. A test article was not predicted to be a skin irritant (GHS No Category) when the mean relative viability of the triplicate tissues was >50%. The mean viability of the test articles was 100.2% ± 11.19, therefore A-1298847.0 is classed as a non-irritant. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11th June 2018 to 7th January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Acute Eye Irritation / Corrosion (405) published by the Ministry of Environmental Protection of Peoples Republic of China in the year 2013

GLP compliance:

yes

Species:

rabbit

Strain:

other: Japanese White

Details on test animals or tissues and environmental conditions:

Source: Qingdao Kangda Biological technology Limited company
A physical check up was carried out on all animals on arrival. Animals were acclimatised for 11 days and housed individually in Room A115. All animals were weighed and marked on the hair. Clinical observations were performed daily. Animals were raised in suspended stainless steel cages.
Temperature: 17 - 23oC
Humidity: 40-70%
Light sequence: 12 hour light / 12 hour dark
Food: Rabbit Maintenance Feed (Beijing keaoxieli Feed Co. Ltd.)
Water: ad libitum
To avoid or minimise pain and distress in ocular safety testing procedures, a local anaesthetic tetracaine hydrochloride 0.5% 1 drop per eye was used 5 minutes prior to test substance application

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1g test item

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

Observations made 1, 24, 48 and 72 hours after treatment.

Number of animals or in vitro replicates:

3

Details on study design:

To investigate if the test material has eye-irritation potential, a sequenced in vivo test in rabbits was conducted.
The test was performed initially using one animal.
The corrosive or severe irritant effect was not observed in the initial test. A confirmatory test was conducted by exposing two additional animals simultaneously at the 24 hours observation point for the initial animal.

Eye Examination before Testing
Approximately 24 hours prior to dosing, both eyes of each animal were examined with a binocular loupe and 2% aqueous sodium fluorescein in the slit lamp. One drop of 2% aqueous sodium fluorescein was placed in the conjunctivae sac of both eyes. After 15 seconds, the eyes were washed using physiological saline. Corneas of both eyes were examined in the slit lamp to see whether there was flourescein detention in the cornea. No eye abnormalities were noted for all treated eyes prior to the test.

Administration Method
0.1g of test item was placed in the conjunctival sac of the right eye of each animal after gently pulling the lower lid away from the eyeball. The lids were gently held together for approximately one second in order to prevent loss of the material. The left eye remained untreated and served as a control.

Observation
Immediately after administration of the test item, the initial reaction was observed and recorded. Animals were observed twice daily for clinical signs and any adverse systemic effects after administration of test item. All treated eyes were examined at approximately 1, 24, 48 and 72 hours after administration. Because all animals had slight irritation, reactions were recorded 24 hours after administration and further observation was made 72 hours after administration. Both eyes of each animal were further examined with the aid of fluorescein sodium and slit lamp. Fluorescein stain was used to aid in scoring at the 24, 48 and 72 hours observation point.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results in the Japanese White Rabbit, E Stage 3 Intermediate could produce reversible eye irritant reactions and was classified as 'unclassified' according to GHS classification criteria for eye irritation

Executive summary:

The study was performed to assess the acute eye irritation/corrosion of E Stage 3 Intermediate in Japanese White rabbits. The method was designed to be in accordance with the guidelines for the testing of chemicals 'Acute Eye Irritation/Corrosion' (405) published by the Ministry of Environmental Protection of People's Republic of China in the year 2013. 

Three male rabbits were used for the study. Each animal was administered 0.1g of the test item in the right eye. The untreated left eye served as the control. The rabbits were observed and recorded for initial reaction immediately, 1, 24, 48 and 72 hours after administration. Mean scores of ocular lesions at approximately 24, 48 and 72 hours after administration were calculated for treated eyes. Individual animal body weights were recorded during the study.

During the initial test, one animal showed iris congestion, swelling, moderate circum corneal hyperaemia, conjunctivae redness and swelling at 1 hour and recovered at 48 hours after administration. During the confirmatory test, the two animals showed conjunctivae redness at approximately 1 hour and recovered at 24 hours after administration. The mean scores of iris and conjunctivae redness were both 0.3.  No abnormal clinical signs were observed and all animals showed expected gain in bodyweight during the study. 

Based on the results in the Japanese White Rabbit, E Stage 3 Intermediate could produce reversible eye irritant reactions and was classified as 'unclassified' according to GHS classification criteria for eye irritation.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th September 2018 to 2nd January 2019

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:

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:

no

Remarks:

The study has been conducted with a reputable lab for other international submission.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test article.
Four corneas were incubated in the presence of the test article at 32 ± 1ºC. Three corneas were incubated in the presence of each control at 32 ± 1ºC.

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The solid test article, A-1298847.0, was administered to the test system as 20% (w/v) (200 mg/mL) dilutions in sterile, deionized water.

Duration of treatment / exposure:

The solid test article A-1298847.0, and the concurrent assay controls (20% (w/v) dilution of imidazole and sterile, deionized water) were exposed to the corneas for 4 hours. After removal of the test or control article from the corneas, a final opacity was determined (i.e., the corneas did not receive a post-exposure incubation).

Number of animals or in vitro replicates:

Four corneas were incubated in the presence of the test article. Three corneas were incubated in the presence of each control.

Details on study design:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test article to isolated bovine corneas. Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test article. An In Vitro Score was determined for the test article based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas.

A-1298847.0 was administered to the test system as 20% (w/v) (200 mg/mL) dilutions in sterile, deionized water.The test article dilution was prepared by weighing the test article into a conical tube, adding sterile, deionized water, until a 20% (w/v) dilution was achieved, and then vortexing the dilution for approximately 1 minute prior to application. The positive control (a 20% (w/v) dilution of imidazole prepared in Complete Minimal Essential Medium (without phenol red)) and the negative control (sterile, deionized water) were tested concurrently.

Four corneas were incubated in the presence of the test article and three in the presence of the control at 32 ± 1ºC .for four hours. After removal of the test or control article from the corneas, a final opacity was determined (i.e., the corneas did not receive a post-exposure incubation).

Irritation parameter:

in vitro irritation score

Value:

2.1

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro score was calculated as 2.1 and as it is ≤3 is classified as GHS no category according to OECD TG 437.

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test article to isolated bovine corneas. Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test article. An In Vitro Score was determined for each test article based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas. The methods and procedures used in this assay were consistent with OECD Test 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 (2017)

Method Synopsis:

A-1298847.0 was administered to the test system as 20% (w/v) (200 mg/mL) dilutions in sterile, deionized water. The test article dilution was prepared by weighing the test article into a conical tube, adding sterile, deionized water, until a 20% (w/v) dilution was achieved, and then vortexing the dilution for approximately 1 minute prior to application. The positive control (a 20% (w/v) dilution of imidazole prepared in Complete Minimal Essential Medium (without phenol red)) and the negative control (sterile, deionized water) were tested concurrently.

Four corneas were incubated in the presence of the test article and three in the presence of the control at 32 ± 1ºC .for four hours. After removal of the test or control article from the corneas, a final opacity was determined (i.e., the corneas did not receive a post-exposure incubation). The opacity value was determined to be -2.3, the OD490 value is -0.012 and the in vitro score calculated as 2.1.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available