5-chloro-1,3-dihydro-1-(4-piperidinyl)-2H-benzimidazol-2-one

Endpoint summary

Description of key information

Skin irritation

In a K2 in vivo skin irritation study in New Zealand White rabbits according to OECD Guideline 404 and EU method B.4, no evidence for skin irritation was noted for T000990.

Eye irritation

In a K1 Bovine Corneal Opacity and Permeability test, performed according to OECD guideline 437 and EU Method B.47, T000990 induced serious eye damage and based on these results should be classified for Eye damage category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations and the criteria of the CLP Regulation (EC) No 1272/2008.

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:

2003-07-01 to 2003-07-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

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:

no

Remarks:

This study was conducted in a facility operating to Good Laboratory Practice within the UK national GLP monitoring programme, but the study report has not been audited by the QA Unit. No formal claim of GLP compliance is made for this study.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS: no data

ENVIRONMENTAL CONDITIONS: no data

IN-LIFE DATES: no data

Type of coverage:

semiocclusive

Preparation of test site:

not specified

Vehicle:

not specified

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g
- Concentration (if solution): not applicable

Duration of treatment / exposure:

Single 4-hour treatment

Observation period:

1, 24, 48, and 72 hours after administration

Number of animals:

3 males

Details on study design:

TEST SITE: no data

REMOVAL OF TEST SUBSTANCE: no data

SCORING SYSTEM:
according to Draize J H (1959)

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: 1, 24, 48 and 72 hours

Score:

ca. 0

Max. score:

8

Reversibility:

fully reversible within: not applicable

Irritation parameter:

erythema score

Basis:

mean

Remarks:

Male number 16

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Remarks:

Male number 17

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Remarks:

Male number 18

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

Male number 16

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

Male number 17

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

Male number 18

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No evidence of skin irritation was reported. All animals received a score of 0 for erythema and edema at 1, 24, 48 and 72 hours.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was reported not to be a skin irritant under the test conditions.

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 2015-06-01 to 2015-06-11

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 Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Principles of method if other than guideline:

The study procedures described in the study are also in compliance with the following documents:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
- Gautheron P, Dukic M, Alix D and Sina J F, Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A14JB3414
- Expiration date of the lot/batch: 2015-10-01 (retest date)
- Purity test date: 2014-12-22


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data

OTHER SPECIFICS:
correction factor : 1

Species:

other: bovine eyes

Strain:

other: not applicable

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. Bovine eyes were used as soon as possible but within 4 hours after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

- Preparation of 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.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) 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°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

- Cornea selection and opacity reading: 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 (OP-KIT, MC2, Clermont-Ferrand, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 3 were not used. Three corneas were selected at random for each treatment group.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

Amount / concentration applied:

TEST MATERIAL
- A 20% (w/w) non-homogenous suspension of the test item was prepared in physiological saline.
- Amount(s) applied (volume or weight with unit): 750 µl

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µl
- Lot/batch no. (if required): no data
- Purity: no data
- Source: Eurovet Animal Health, Bladel, The Netherlands

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): 20% (w/v)

Duration of treatment / exposure:

Corneas were incubated for 240 ± 10 minutes at 32 ± 1°C.

Observation period (in vivo):

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 (Eagle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Details on study design:

OPACITY MEASUREMENT
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. 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 positive control or test item treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test item 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 (Merck) 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 (Sigma-Aldrich Chemie GmbH, Germany). 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 (TECAN Infinite® M200 Pro Plate 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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

INTERPRETATION
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

74.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: 70.3 to 80.6

Irritation parameter:

cornea opacity score

Run / experiment:

2

Value:

62.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: 56 to 73

Irritation parameter:

other: Permeability score

Run / experiment:

2

Value:

0.791

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: 0.509 to 0.952

Other effects / acceptance of results:

First test:
- The individual opacity scores for the negative controls ranged from 4 to 8, which is above the historical range, therefore the test was cancelled.

Second test:
- The individual in vitro irritancy scores for the negative controls were 0.0. The individual positive control in vitro irritancy scores ranged from 116.2 to 156.2. The in vitro irritancy scores ranged from 70.3 to 80.6 after 240 minutes of treatment with the test item.
- The corneas treated with the test item showed opacity values ranging from 56.0 to 73.0 and permeability values ranging from 0.509 to 0.952. The corneas were turbid after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium.
- The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 133.6 (116.2 to 156.2) and within the historical positive control data range. Furthermore the opacity and permeability values of the positive control were within two standard deviations of the current historical mean. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
- The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Since the test item induced an IVIS ≥ 55, it is concluded that the test item induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Sanders (2004) investigated acute dermal irritation of T000990 in New Zealand White rabbits (3 males after 4 hours of exposure to 0,5 g of test item). Skin reactions were recorded 1, 24, 48 and 72 hours after administration and scored according to the Draize scale.

No evidence of skin irritation was noted. The test item did not meet the criteria for classification as irritant or corrosive according to the criteria of the CLP Regulation (EC) No 1272/2008.

An in vitro skin irritation study was waived based on the justification that adequate data from an in vivo skin irritation study are available.

Eye irritation:

Eurlings (2015) investigated eye irritation in an in vitro bovine corneal opacity-permeability (BCOP) assay. 750 µl of T000990 (20% w/w non-homogenous suspension in physiological saline) was applied for 240 minutes. Both opacity and permeability were measured and the resulting objective values were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The corneas treated with the test item were turbid after 240 minutes treatment. A mean in vitro irritancy score of 74.5 (70.3 to 80.6) was calculated after 240 minutes of treatment. Since the test item induced an IVIS ≥ 55, it is concluded that T000990 induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in the report and should be classified category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

 

In addition, a rabbit enucleated eye test was performed by Sanders (2004) to assess the ocular irritancy potential of T000990. Three enucleated eyes of New Zealand White rabbits were treated with 0.1 mL of T000990. Corneal opacity (60, 120, 180 and 240 minutes after application), corneal swelling (60, 120 and 240 minutes after application) and fluorescein uptake (240 minutes after application) were observed and scored. No indication of irritation was noted. The test item was considered unlikely to have the potential to cause severe ocular irritancy in-vivo.

The BCOP study is considered the key result for assessing the eye irritation endpoint. According to Chapter R.7a: Endpoint specific guidance Version 5.0 - December 2016 (R.7.2.11.2), data obtained from non-validated suitable in vitro tests can only be used according to the criteria set out in section 1.4 of Annex XI to the REACH Regulation, i.e. only positive results can be accepted in a weight of evidence approach. As the result of the non-validated REET test was negative, this study was added to the dossier as supporting evidence and the newly conducted and validated BCOP study is selected as key study for classification purposes.

Justification for classification or non-classification

Skin irritation:

According to the in vivo acute dermal irritation study no evidence for skin irritation was noted for T000990. The test item did not meet the criteria for classification as irritant or corrosive according to the criteria of the CLP Regulation (EC) No 1272/2008.

Eye irritation:

According to the in vitro eye irritation study (BCOP), T000990 should be classified for Eye damage category 1.