N-{[Benzyl(ethyl)amino]-[(chloro-cyano-nitrophenyl)substituted]phenyl}acetamide

Endpoint summary

Description of key information

FAT 41047/A was considered to be non-irritating to skin and eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Source: In Vivo Bio Sciences. Shed No.23, Assessment No.154, Kodigehalli Village,Magadi Road, Bangalore - 560091 Karnataka, India.

Age at treatment : 3 months

Body weight range at treatment : 2.2895 to 2.3708 kgs

Acclimatization : One rabbit (RBa4287) was acclimatized for six days while the other two (RBa4288 and RBa4289) rabbits were acclimatized for seven days under laboratory conditions after physical examination.

Housing : The rabbits were housed individually in rabbit cages (LxBxH 65 x 65 x 45 cm) with noryl shallow cage body and facilities for pelletted food (Stainless steel feed hopper) and drinking water (750 mL markrolon bottle fitted with sipper tube). The litter collection trays (noryl waste tray) were changed daily (except on Sundays). The water bottle and feed hopper were changed once a week. The noryl shallow cage body and the rack were washed once in 15 days.

Diet: ad libitum
The animals were offered rabbit feed manufactured Krishna Valley Agro Tech LLP, MIDC Kupwad block, Sangli, Maharashtra.

Water: ad libitum
The animals were provided deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India.

Environmental Conditions :
Animals were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (13.2 air changes/hour). Temperature : 21 to 23°C, relative humidity : 65 to 67% with a 12 hour light and 12 hour dark cycle.
The maximum and minimum temperature and relative humidity in the experimental room were recorded once daily. The relative humidity in the experimental room was calculated from dry and wet bulb temperature recordings.

IN-LIFE DATES : 24 July 2018 to 30 July 2018.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 500 mg of test item was added to 1 mL of Milli-Q water to form paste.

Negative control : Milli-Q water

VEHICLE
- The test substance was moistened with distilled water, immediately before application, to ensure close contact to the animal's skin.

Duration of treatment / exposure:

4 h

Observation period:

Up to 72 h (The skin reactions were assessed at approximately 45 min, 24, 48 and 72 h after the removal of the dressings and test substance)

Number of animals:

Three male animals.

Details on study design:

TEST SITE
- Area of exposure: Approx. 6 cm²
- Type of wrap if used:The patches were held in place by a strip of adhesive tape and crepe elastic bandage wound around the torso, which was anchored using safety pins.

REMOVAL OF TEST SUBSTANCE
- Washing : Removed at the end of the exposure period using a tissue moistened with tap water and subsequently.
- Time after start of exposure: 4 h

SCORING SYSTEM: Draize scoring system.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other:

Remarks:

black colour imparted by the test item on the treated area on 24hr observation period only.

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

other:

Remarks:

black colour imparted by the test item on the treated area on 24hr observation period only.

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No corrosion or skin irritation was observed during the study.

Other effects:

Colouration: Black staining of the treated skin by the test substance was observed for animal #1 for time point of 24h.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was considered to be a non-irritant to skin.

Executive summary:

A study was performed to assess the skin irritation potential of the FAT 41047/A (of ca. 93.09 % purity) in New Zealand White rabbits according to OECD Guideline 404 in compliance with GLP.

500 mg of test substance was dissolved in 1mL of Milli-Q water to form a paste which was applied to clipped skin area of each animal under a 2 x 3 sq.cm gauze pad. The pad was allowed to remain in contact with the skin for 4 h. Animals were scored for irritation reactions after 60 minutes, 24h, 48h and 72 h respectively. All test animals were monitored daily for viability, mortality and clinical signs. Body weights were recorded pre-test and at study termination.

Black staining of the treated skin by the test substance was observed, which made scoring of erythema difficult at 60 minutes and 24 h in 1/3 animals. No erythema or oedema was observed for skin irritation at 1/24/48/72 hrs. The erythema and edema score was zero at all time points. No signs of body weight variability, toxicity or mortality were observed in any animal during the study period.

Based on the findings of this study, the test substance was considered to be non-irritating to skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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)

Test system:

human skin model

Source species:

human

Justification for test system used:

The assessment of skin irritation typically involved used of laboratory animals according to OECD 404. To avoid pain and suffering of animals, OECD 439 was introduced which is an invitro model for skin irritation. OECD 439 uses reconstructed human epidermis (RhE) which closely mimics biochemical and physiological properties of the upper parts of the human skin, ie the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 20 - 28 °C
- Temperature of post-treatment incubation: Room temperature

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 42h
- Spectrophotometer: TECAN Infinite® M200 Microplate Reader
- Wavelength: 570nm

NUMBER OF REPLICATE TISSUES: Triplicate

PREDICTION MODEL / DECISION CRITERIA:
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.]

Amount/concentration applied:

10 mg/tissue of test substance was applied topically to the EpiSkin three dimensional RhE model.

Negative control : 10 microliter of Phosphated Buffered Saline (PBS).

Positive control : 10 microliter of Sodium Dodecyl Sulfate (SDS)

Duration of treatment / exposure:

Incubation for 15 minutes at room temperature (20 -28 degree celcius)

Duration of post-treatment incubation (if applicable):

Tissues were incubated with MTT solution for 4 hours and then extracted with acidified isopropanol to quantify spectrophotometrically at wavelength of 570 nm.

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

R1

Value:

ca. 437.34

Positive controls validity:

valid

Remarks:

9.63

Remarks on result:

not determinable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

R2

Value:

ca. 442.82

Positive controls validity:

valid

Remarks:

9.69

Remarks on result:

not determinable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

R3

Value:

ca. 438.37

Positive controls validity:

valid

Remarks:

9.51

Remarks on result:

not determinable

Other effects / acceptance of results:

True tissue viability is not calculated in this study because the %NSCliving + NSMTT is > 30%

	% Individual Viability
	R1	R2	R3	Mean	SD
Positive control	9.63	9.69	9.51	9.61	0.09
Test item	437.34	442.82	438.37	439.51	2.91
% NSC living tissue	250.18	251.58	-	250.88	0.99
% NSMTT killed tissue	3.74	3.63	-	3.69	0.078
% NSC killed tissue color	17.86	15.37	-	16.62	1.76

Interpretation of results:

study cannot be used for classification

Conclusions:

The viability of the test item was 439.51%. Since, the sum of %NSMTT and %NSCliving was >30% relative to the negative control, the final viability is not applicable to draw a conclusion.

Executive summary:

The test item, FAT 41047/A was tested for its possible skin irritation potential using a three-dimensional Reconstructed Human Epidermis model EpiSkin through topical application for 15 minutes according to OECD test guideline 439.

The test item is a powder and was directly applied on the top of the skin tissues at 10 mg/tissue along with 5 microliters of negative control (PBS) and exposed for 15 mins. Ten microlitres of PBS and 10 microlitres of 5% SDS were used as the negative and positive controls, respectively.

In the preliminary tests, FAT 41047 was identified as producing direct MTT reduction and color interference and thus adapted controls evaluation was performed.

After approximately 42 hour post incubation period, irritation potential was evaluated by assessing the cytotoxic (irritancy) effect. The cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured on formazan production from MTT and the end of treatment.

Skin irritation is expressed as the ability of the test item to reduce the cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 mins of treatment with the test item is compared with negative control tissues.

The absolute mean Optical density (OD) at 570 nm of negative control was 0.6421. The positive control had a mean cell viability of 9.61% after 15 mins exposure, indicating the test system functioned properly.

The viability of the test item was 439.51%. Since, the sum of %NSMTT and %NSCliving was > 30% relative to the negative control, the final viability is not applicable to draw a conclusion.

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:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Eurofins Advinus Limited, Bengaluru-560058
- Age at study initiation: 5-6 months
- Weight at study initiation: 2.6377 to 2.7593 kg
- Housing: The rabbits were housed individually in rabbit cages (approx. size: L 65 x B 65 xH 45 cm) with noryl shallow cage body and facilities for pelleted food (Stainless steel feed hopper) and drinking water (750 mL markrolon bottle fitted with sipper tube). The perforated noryl raised shelf for enrichment was provided. The litter collection trays (noryl waste tray) were changed daily (except on Sunday). The water bottles and feed hoppers were changed once a week.

- Diet: The animals were offered rabbit feed manufactured Krishna Valley Agrotech LLP, MIDC Kupwad block, Sangli, Maharashtra.
- Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India, was provided to animals in markrolon bottles with stainless steel sipper tubes.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23
- Humidity (%): 65 to 67
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark cycle.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

100 mg

Observation period (in vivo):

72h

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:
CORNEA
A: Opacity and degree of density (most dense area scored)
0 - No opacity
1 - Scattered or diffuse area, details of iris clearly visible
2 - Easily discernible translucent areas, details of iris slightly obscured
3 - Nacreous area; no details of iris visible; size of pupil barely discernible
4 - Opaque cornea; iris not discernible through the opacity

B: Area of cornea involved
1 - One quarter (or less) but not zero
2 - Greater than one quarter, but less than half
3 - Greater than half, but less than three quarters
4 - Greater than three quarters, up to whole area

IRIS
A: Values
0 - Normal
1 - Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to light (a sluggish reaction is considered to be an effect
2 - Haemorrhage, gross destruction, or no reaction to light


CONJUNCTIVAE
A: Redness (refers to palpebral and bulbar conjunctiva excluding cornea and iris)
0 - Vessels normal
1 - Some blood vessels hyperaemic (injected)
2 - More diffuse, deeper crimson red, individual vessels not easily discernible
3 - Diffuse beefy red

B: Chemosis
0 - No swelling
1 - Any swelling above normal (includes nictitating membrane)
2 - Obvious swelling with partial eversion of lids
3 - Swelling with lids about half closed
4 - Swelling with lids about half closed to completely closed

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Other effects:

There were no clinical signs of toxicity and pre-terminal deaths.
The body weights of all rabbits increased during the observation period.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the present study results the test item FAT 41047/A TE is “Non-Irritant” on the eye of New Zealand white rabbits.

Executive summary:

The acute eye irritation / corrosion study in New Zealand White rabbits was conducted to evaluate the eye irritation / corrosion potential of FAT 41047/A TE. This study was conducted in accordance with OECD 405 test guideline in a GLP compliant lab.

A single dose of the test item was instilled in to the conjunctival sac of the rabbit’s eye.

On test day one (treatment day), a quantity of 100 mg of finely ground test item was instilled into the conjunctival sac of the left eye of the animal after gently pulling the lower lid away from the eyeball. The lids were then held together gently for about one second to prevent loss of test item. The right eye remained untreated and served as the reference control. All the rabbits were treated in a similar manner.

The eyes of each rabbit were examined at 1, 24, 48 and 72 hours post-instillation and scored.

Mean corneal opacity, iris, conjuctival redness, chemosis score was 0. No irritation was observed. There were no clinical signs of toxicity and pre-terminal deaths and no abnormality was detected at necropsy in any of the animals.

Based on the present study results the test item FAT 41047/A TE is “Non-Irritant” on the eye of New Zealand white rabbits according to Regulation (EC) No 1272/2008 of the European Parliament and of the council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, as there were no eye reactions in any of the treated rabbits.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

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

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine eye

Details on test animals or tissues and environmental conditions:

The bovine eyes were procured from local abattoir and transported in a jar containing Hank’s Balanced Salt Solution (HBSS) and penicillin-streptomycin (100 IU & 100 μg/mL) in an ice box.

Post pretest examination, selected eyes were dissected to isolate the corneas from surrounding tissue and then placed in a container with fresh HBSS (Hank’s Balanced Salt Solution).

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

Amount / concentration applied:

100 mg of test item per cornea

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

90 mins

Number of animals or in vitro replicates:

Triplicate

Details on study design:

Bovine eyes were procured from local abattoir and transported in a jar containing Hank’s Balanced Salt Solution (HBSS) and penicillin-streptomycin (100 IU & 100 µg/mL) in an ice box.

Immediately after receiving the eyes in the lab, all eyes were observed for any evidence of vascularization, pigmentation, opacity or scratches. Selected eyes were dissected to isolate the corneas from surrounding tissue and then placed in a container with fresh HBSS.

Isolated corneas were mounted on the cornea holder. The cornea was projected into the anterior chamber. The EMEM without phenol red was added in to both the chambers and kept in an incubator (32°C) for 1 hour.

The blank value I0 (lux) was obtained with each of 30 available cornea holders containing medium but without cornea. The mean of the blank value obtained was 502 lux (I0). This value was considered while calculating the initial opacity and final opacity readings.

After pre-test incubation, the EMEM from both the chambers was removed and refilled with the fresh EMEM. The pre-treatment cornea reading (lux) was measured and initial opacity was calculated for all corneas.
The initial opacity was calculated. The corneas which exhibited initial opacity < 7 were considered for further experiment.

NUMBER OF REPLICATES : Three for each test group.

NEGATIVE CONTROL : Distilled water

POSITIVE CONTROL : Chlorhexidine

100 mg of test substance per cornea, 0.75 mL negative control and enough quantity of positive control to cover the entire corneal surface were applied undiluted respectively. The test substance was exposed for four hours in incubation medium at 32 (±1) ºC in an open chamber.

POST INCUBATION PERIOD : no

After Incubation period, the anterior chamber was rinsed with EMEM without phenol red. Cornea's were incubated at 32 (±1) °C with 1 mL of 5 mg/mL sodium fluorescein solution (in Dulbecco’s Phosphate-Buffered Saline) for 90 minutes.

METHODS FOR MEASURED ENDPOINTS :
Final opacity of all corneas was measured with fresh EMEM using an OP3.0 Opacitometer. Permeability was measured using Microplate reader/Spectrophotometer used to measure permeability. (Make: Tecan, Model: Infinite M200.)

IVIS (In Vitro Irritancy Score) was calculated using Mean Opacity value and Mean Permeability value.

Irritation parameter:

in vitro irritation score

Value:

3.111

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Other effects / acceptance of results:

The corneas treated with distilled water exhibited IVIS score of 0.9235.

The corneas treated with positive control (Chlorhexidine) exhibited IVIS score of 170.8765.

Calculation of In Vitro Irritation Score (IVIS*)

Treatment group	Mean opacity value	Mean Permeability value	IVIS	Classification
Negative control (Distilled water)	0.67	0.00169	0.9235	No category
Chlorhexidine	169	0.1251	170.8765	Category 1
FAT 41047/A TE	3.66	-0.0366	3.111	No predication can be made

*IVIS = Mean Opacity value + (15 × Mean Permeability value)

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item FAT 41047/A TE exhibited an IVIS score of 3.111. From the results, no predictions could be made that the test item causes eye irritation/serious eye damage.

Executive summary:

The evaluation of the occular irritancy of FAT 41047/A TE was carried out according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability) test.

The test substance, negative control and positive control were applied undiluted on the corneal surface by means of open chamber method. The control and treated corneas were then subjected to the opacity and permeability measurements.

The negative control (Distilled water) reported IVIS score 0.9235 and the positive control (Chlorhexidine) reported IVIS score 170.8765. The test item FAT 41047/A exhibited an IVIS score of 3.111 and was classified under ‘No prediction can be made’ as per UN GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

FAT 41047/A was tested for its possible skin irritation potential using a three-dimensional Reconstructed Human Epidermis model EpiSkin through topical application according to OECD test guideline 439. The viability of the test item was 439.51%. Since, the sum of %NSMTT and %NSCliving was > 30% relative to the negative control, the final viability is not applicable to draw a conclusion.

Hence an in vivo study was performed to assess the skin irritation potential of the FAT 41047/A in New Zealand White rabbits according to OECD Guideline 404. No erythema or edema was observed after exposure during the study. The test substance was considered to be non-irritating to skin.

Eye irritation:

The evaluation of the occular irritancy of FAT 41047/A TE was carried out in a bovine corneal opacity study conducted according to OECD Guideline 437. The negative control (Distilled water) reported IVIS score 0.9235 and the positive control (Chlorhexidine) reported IVIS score 170.8765. FAT 41047/A exhibited an IVIS score of 3.111 and was classified under ‘No prediction can be made’ as per UN GHS.

Hence, an in vivo study was performed to assess the acute eye irritation / corrosion potential of FAT 41047/A in New Zealand White rabbits in accordance with OECD 405 test guideline. No irritation was observed at any observation points. Thus, FAT 41047/A TE was found to be “Non-Irritant” to the rabbit eye.

Justification for classification or non-classification

FAT 41047/A is considered to be not irritating to the skin and eyes, hence it does not warrant classification according to the criteria of Regulation (EC) No. 1272/2008.