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Description of key information

Skin irritation / corrosion:


In vitro: The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour on a human three dimensional epidermal model (EpiDerm (EPI-200)) based on OECD 431.


The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.


In vivo: Read across to Sarolaner API


A primary dermal irritation study was conducted on three albino rabbits using test item in accordance with OECD 404. The erythema and edema scores for each animal are all 0 for 24, 48, and 72 h observations. The test item is rated non-irritating.


 


Eye irritation:


The eye damage of the test item was tested through topical application for approximately 240 minutes in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD 437.


Since the test item induced an IVIS ≤ 3 in 5 out of 6 corneas, no classification is required for eye irritation or serious eye damage.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2012-09-25 to 2012-09-28
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)
Specific details on test material used for the study:
Lot No.: PF-06450567-00-0022
Purity: Not provided
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rich-Glo II, Inc; Wills Point, TX
- Age at study initiation: ca. 3 months
- Weight at study initiation: 2.229-2.824 kg
- Housing: Suspended, wire bottom, stainless steel, 1 per cage
- Diet: PMI Feeds, ca. 8 oz. daily
- Water: Municipal water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 49-89
- Air changes (per hr): 10-12 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

IN-LIFE DATES: From: 25 Sep 12 To: 28 Sep 12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
water
Controls:
yes, concurrent no treatment
Amount / concentration applied:
500 mg of test item moistened with 0.5 mL of DI water
Duration of treatment / exposure:
4 hous
Observation period:
72 hours
Number of animals:
1 male, 2 females (nulliparous & non-pregnant)
Details on study design:
TEST ITEM ADMINISTRATION
On Day 0, 500 mg of test item moistened with 0.5 mL of DI water was applied to each test site and covered with a 4 ply, 2.5 x 2.5 cm surgical gauze patch. Each patch was secured in place with a strip of non-irritating adhesive tape. The entire trunk of each animal was loosely wrapped with a semi-permeable dressing (orthopedic stockinette) which was secured on both edges with strips of tape to retard evaporation of volatile substances and to prevent possible ingestion of the test item.

REMOVAL OF TEST SUBSTANCE
- Washing: The test sites were gently washed with room temperature tap water and a clean cloth to remove as much residual test item as possible.
- Time after start of exposure: 4 h

OBSERVATION TIME POINTS
The test sites were observed for erythema and edema formation, and any other dermal effects at 1, 24, 48 and 72 hours after unwrap.

SCORING SYSTEM:
- Method of calculation: Draize
Irritation parameter:
erythema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Irritation parameter:
erythema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Irritation parameter:
edema score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Irritant / corrosive response data:
Erythema and edema were not observed at any time throughout the study. No other dermal effects were observed during the study.

Individual dermal irritation scores:














































Animal No.



Erythema score



Edema score



24 h



48 h



72 h



24 h



48 h



72 h



1



0



0



0



0



0



0



2



0



0



0



0



0



0



3



0



0



0



0



0



0


Interpretation of results:
GHS criteria not met
Conclusions:
The primary irritation index of 0.0 out of a possible 8.0 was obtained from the observations at 1, 24, 48 and 72 hours, and was used to give test item a descriptive rating of non-irritating.
Executive summary:

A primary dermal irritation study was conducted on three albino rabbits using test item in accordance with OECD 404.


There was one intact test site per animal. Each test site was treated with 500 mg of test item moistened with 0.5 mL of DI water and covered with a semi-permeable dressing. The test item was maintained in contact with the skin for 4 hours. Observations for dermal irritation and defects were made at 1, 24, 48 and 72 hours after removal of the dressings.


The erythema and edema scores for each animal are all 0 for 24, 48, and 72 h observations.


The test item is rated non-irritating.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2020-12-14 to 2020-12-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
18 June 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: 465884
Purity: 100%
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 33783 Kit F
- Certificate of Analysis issue data: 2020-12-16
- Date of initiation of testing: 2020-12-14

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes exposure: room temperature, 1-hour exposure: 37.0 ± 1.0°C (actual range 35.9 - 37.3°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if 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 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%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
29.50 to 46.94 mg
Duration of treatment / exposure:
3 minutes, 1 hour
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1-hour exposure
Value:
99
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3-minute exposure
Value:
91
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0007 and 0.0058, respectively. Therefore it was concluded that the test item did not induce color interference.
In addition, because no color change was observed in the presence of MTT it was concluded that the test item did not interact with the MTT endpoint.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.2%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤14%, indicating that the test system functioned properly.
Interpretation of results:
other: Not corrosive
Conclusions:
The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate test item for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour based on OECD 431.


Skin tissue was moistened with 25 µL of Milli-Q water and at least 25 mg of the test item was applied directly on top of the skin tissue.


The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 91% and 99%, respectively. Because the mean relative tissue viability for the test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test item is considered to be not corrosive.


In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not 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:
From 2020-11-24 to 2020-12-15
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)
Version / remarks:
26 June 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Batch No.: 465884
Purity: 100%
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.
- Selection and preparation of corneas:
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32±1℃. The corneas were incubated for the minimum of 1 hour at 32±1℃.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.
- Quality check of the isolated corneas:
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
300.1 to 338.0 mg
Duration of treatment / exposure:
240 ± 10 mins
Number of animals or in vitro replicates:
3
Details on study design:
NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: physiological saline

POSITIVE CONTROL USED: 20% (w/v) Imidazole

APPLICATION DOSE AND EXPOSURE TIME:
The medium from the anterior compartment was removed and 750 μL of the negative control
and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of
the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a
way that the cornea was completely covered (300.1 to 338.0 mg). The holder was slightly
rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of
the solutions over the entire cornea. Corneas were incubated in a horizontal position for
240 ± 10 minutes at 32±1℃.

REMOVAL OF TEST SUBSTANCE:
After the incubation the solutions and the test item were removed and the epithelium was washed at least three times with MEM with phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (OD490)
- Others: Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

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:
The second experiment
Value:
>= -2.5 - <= -0.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
In the first experiment, the individual in vitro irritancy scores for the negative controls ranged from -1.4 to 0.3. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 112 to 150. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test item showed opacity values ranging from 0.4 to 6.5 and permeability values ranging from -0.006 to 0.001. The corneas were translucent after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 0.3 to 6.4 after 240 minutes of treatment with the test item. The mean in vitro irritancy score was 2.5 after 4 hours of treatment with the test item. Since the results were spread over 2 categories (IVIS of 0.3, 6.4 and 0.9, respectively), the test was inconclusive and a repeat experiment was performed.
In the second experiment, the individual in vitro irritancy scores for the negative controls ranged from 0.6 to 2.8. The corneas treated with the negative control item were clear after the 240 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 106 to 119. The corneas treated with the positive control were turbid after the 240 minutes of treatment. The corneas treated with the test item showed opacity values ranging from -2.8 to -1.1 and permeability values ranging from 0.012 to 0.015. The corneas were clear after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.5 to -0.9 after 240 minutes of treatment with the test item.
Interpretation of results:
GHS criteria not met
Conclusions:
Since the test item induced an IVIS ≤ 3 in 5 out of 6 corneas, no classification is required for eye irritation or serious eye damage.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test) according to OECD 437. The eye damage of the test item was tested through topical application for approximately 240 minutes.


In the first experiment, the in vitro irritancy scores ranged from 0.3 to 6.4 after 240 minutes of treatment with the test item. The mean in vitro irritancy score was 2.5 after 4 hours of treatment with the test item. Since the results were spread over 2 categories (IVIS of 0.3, 6.4 and 0.9, respectively), the test was inconclusive and a repeat experiment was performed.


In the second experiment, the in vitro irritancy scores ranged from -2.5 to -0.9 after 240 minutes of treatment with the test item.


Since the test item induced an IVIS ≤ 3 in 5 out of 6 corneas, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Skin irritation / corrosion:


In vitro skin corrosion, OECD 431: Not corrosive


In vivo skin irritation, Read across to Sarolaner API, OECD 404:


Mean score for erythema at 24, 48, 72 h for 3 tested animal: 0, 0, 0


Mean score for edema at 24, 48, 72 h for 3 tested animal: 0, 0, 0


According to Regulation (EC) No 1272/2008, table 3.2.2, this substance should not be classified for this endpoint.


 


Eye irritation:


In vitro eye irritation, key study, OECD 437: the in vitro irritancy scores < 3 (ranged from -2.5 to -0.9) after 240 minutes of treatment with the test item.


According to Regulation (EC) No 1272/2008, table 3.3.2, this substance should not be classified for this endpoint.