Imidodicarbonic acid, 2-[5-bromo-3-[(1R)-1-[2-[[[(5-cyano-1-methyl-1H-pyrazol-3-yl)methyl]methylamino]carbonyl]-5-fluorophenyl]ethoxy]-2-pyridinyl]-, 1,3-bis(1,1-dimethylethyl) ester, compd. with 2-methyl-2-butanol (1:1)

Endpoint summary

Description of key information

Skin irritation:
Two studies are available:
1) An acute dermal irritation study (Latour J.E.H.M., MSc., 2016) is available which is key study. This study showed that the test substance is not irritating.
2) An in vitro skin corrosion study (Eurlings I.M.J., PhD., 2016) is available which is supporting study. This study showed that the test substance is not corrosive.
Eye irritation:
One in vitro study is available (Eurlings I.M.J., PhD., 2016) which is key study. This study showed that the test substance is not corrosive and not irritating to bovine eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 Mar to 08 Apr 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: between 12 and 24 weeks old
- Weight at study initiation: at least 1.5 kg
- Housing: Animals were individually housed in labeled cages with perforated floors and shelters.
- Diet (e.g. ad libitum): Pelleted diet for rabbits approximately 100 grams per day. Hay and wooden sticks were available during the study period.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): a 12-hour light/12-hour dark cycle

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: watery ethanol (50% v/v)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 grams of the test substance

VEHICLE
- Amount(s) applied (volume or weight with unit): 1 mL

Duration of treatment / exposure:

4 h

Observation period:

72 h

Number of animals:

3 males

Details on study design:

TEST SITE
- Area of exposure: approximately 150 square centimeters (10x15 cm)
- Type of wrap if used: metalline patch mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): tap water
- Time after start of exposure: Four hours

OBSERVATION:
-Mortality/Viability: Twice daily.
-Toxicity: At least once daily.
-Body Weight: Day of treatment (prior to application) and on the day of the final observation
-Irritation: The skin reactions were assessed at approximately 1, 24, 48 and 72 hours after the removal of the dressings and test substance. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of the untreated skin of each animal served as controls.
-Necropsy: No necropsy was performed according to study plan.

SCORING SYSTEM: Draize

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

mean

Time point:

other: Mean of 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritant / corrosive response data:

No skin irritation was caused by 4 hours exposure to test substance.
There was no evidence of a corrosive effect on the skin.

Other effects:

No staining of the treated skin by the test substance was observed and no test substance remnants were seen.
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual skin irritation scores

Animal	41		45		48
Time after exposure	Erythema (0-4)	Oedema (0-4)	Erythema (0-4)	Oedema (0-4)	Erythema (0-4)	Oedema (0-4)
1 hour	0	0	0	0	0	0
24 hours	0	0	0	0	0	0
48 hours	0	0	0	0	0	0
72 hours	0	0	0	0	0	0

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on these results the test substance does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

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 18 to 19 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes

Species:

other: cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), 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.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 311.9 to 340.6 mg

Duration of treatment / exposure:

240±10 minutes

Number of animals or in vitro replicates:

Three eyes each group

Details on study design:

Removal of test substance
- Washing (if done): After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red.
- Time after start of exposure: 240±10 minutes
Reference items
- Negative control: A negative control, physiological saline was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
- Positive control: 20% (w/v) Imidazole solution prepared in physiological saline.
Opacity measurement
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.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.
Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 μL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader. Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 240 minutes

Score:

1.9

Max. score:

2.6

Remarks on result:

other: Scoring based upon IVIS value

Irritant / corrosive response data:

The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 0.9. The individual positive control in vitro irritancy scores ranged from 101 to 154. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
The corneas treated with the test substance showed opacity values ranging from 1.3 to 2.1 and permeability values ranging from 0.009 to 0.031. The corneas were clear after the 240 minutes of treatment with test substance. Hence, the in vitro irritancy scores ranged from 1.4 to 2.6 after 240 minutes of treatment with test substance.

Other effects:

No pH effect of the test item was observed on the rinsing medium.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.9 after 240 minutes of treatment.
Since test substance induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation/corrosion

Two studies are available:

1) An acute dermal irritation study was conducted according to OECD 404 using rabbits (Latour J.E.H.M., MSc., 2016). Key study.

This study showed that the test substance is not irritating.

2) An in vitro skin corrosion study was conducted according to OECD 431 using a human skin model (Eurlings I.M.J., PhD., 2016). Supporting study.

This study showed that the test substance is not corrosive.

Eye irritation:

One in vitro study is available which was conducted according to OECD Guideline 431 under GLP (Eurlings I.M.J., PhD., 2016). Key study.

This study showed that the test substance is not corrosive and not irritating to bovine eyes.

Justification for selection of skin irritation / corrosion endpoint:
Study run to a method comparable with current guidelines and to GLP

Justification for selection of eye irritation endpoint:
Study run to a method comparable with current guidelines and to GLP

Justification for classification or non-classification

Skin irritation/corrosion: Mean scores at 24, 48 and 72 hours for erythema were < 2.3 (actual value 0) and for edema were < 2.3 (actual value 0).

 

Serious eye damage/eye irritation: An in vitro study data is available with IVIS <3 (actual value 1.9).

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.2.2 and section 3.3.2, this substance should not be classified for skin irritation/corrosion and serious eye damage endpoint. In the absence of in-vivo data no definitive conclusion can be drawn on eye irritation endpoint.