cis-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-ylmethyl methanesulphonate monohydrochloride

Endpoint summary

Description of key information

Skin irritation:

In a K2 in vivo skin irritation study in New Zealand White rabbits according to a method similar to OECD Guideline 404 and EU Method B.4, T001202 was observed to be non-irritating to the skin.

Eye irritation:

In a K1 Bovine Corneal Opacity and Permeability (BCOP) test, performed according to OECD guideline 437 and EU method B.47, T001202 induced serious eye damage and based on these results, it 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

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-02-03 to 2004-02-06

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similar to OECD Guideline 404 (Acute Dermal Irritation / Corrosion) and EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion); however, the test substance is not adequately characterized and insufficient information is provided on the test animals, testing methodology and results for a thorough assessment of reliability.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

Inadequate documentation of test material, environmental conditions, animals, methods and results.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

Inadequate documentation of test material, environmental conditions, animals, methods and results.

GLP compliance:

no

Remarks:

The 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 the study.

Specific details on test material used for the study:

No data

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:

0.5 g

Duration of treatment / exposure:

4 hours

Observation period:

72 hours, with observations at 1, 24, 48, and 72 hours

Number of animals:

3

Details on study design:

TEST SITE: no data

REMOVAL OF TEST SUBSTANCE: no data

SCORING SYSTEM:
Draize System 0-8

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal numbers 109, 129 and 133

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal numbers 109, 129 and 133

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Irritant / corrosive response data:

No evidence of erythema/eschar formation or edema was noted at any time at any test site.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was found to be non-irritating to rabbit skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2015-06-01 to 2015-06-02

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: I14KB4717
- Expiration date of the lot/batch: 2016-11-25 (retest date)
- Purity test date: no data
- Certificate of analysis release date: 2014-12-13

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
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A 20% (w/v) suspension of the test item was prepared in physiological saline

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 positive control

yes, concurrent negative control

Amount / concentration applied:

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl

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

Duration of treatment / exposure:

240 ± 10 minutes

Duration of post- treatment incubation (in vitro):

After 240 ± 10 minutes of treatment, opacity was measured with an opacitometer. The permeability measurement of the corneas was performed after the incubation period of 90 minutes ± 5 minutes following the opacity measurement..

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Details on study design:

NEGATIVE CONTROL USED
physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

POSITIVE CONTROL USED
20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 μl of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) test item was introduced onto the epithelium of the cornea. 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°C

REMOVAL OF TEST SUBSTANCE
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.

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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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

Value:

89.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Range: 58.9 to 111.5

Irritation parameter:

cornea opacity score

Value:

86.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: 56.3 to 107.3

Irritation parameter:

other: permeability score

Value:

0.712

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: 0.072 to 0.275

Other effects / acceptance of results:

NEGATIVE CONTROL
- IVIS: 0.0 (-0.7 to 1.4)
- Opacity score: 0.0 (-0.7 to 1.3)
- Permeability score: 0.000 (-0.004 to 0.004)

POSITIVE CONTROL
- IVIS: 134.6 (130.6 to 137.8)
- Opacity score: 115.3 (109.3 to 121.3)
- Permeability score: 1.286 (1.015 to 1.744)

OTHER EFFECTS
The corneas were translucent after the 240 minutes of treatment with the test item. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed.

ACCEPTANCE OF RESULTS
- 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.
- The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 134.6 (130.6 to 137.8) 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.
- 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:

The test item induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 89.2 (58.9 to 111.5) after 240 minutes of treatment.
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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2004-01-26 to 2004-09-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Only a study summary was available for review which provided limited details on the test substance and methodology; however, sufficient information was provided to deem the study reliable with restrictions.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The ocular irritancy potential of the test material was assessed using the Rabbit Enucleated Eye Test (REET). This method involved the application of the test material onto the cornea of the enucleated eye.

GLP compliance:

no

Remarks:

This study was conducted in facility operating to GLP 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.

Specific details on test material used for the study:

no data

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Test animales:
- Five enucleated eyes, obtained from the New zealnd white strain of rabbit.

Environmental conidtions:
- The eyes were maintained at a temeprature of 32°C +/-1.5°C within the superfusion apparatus.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL (approx. 55 mg)
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): no data
- Concentration (if solution): 0.9% sodium chloride

Duration of treatment / exposure:

single application

Number of animals or in vitro replicates:

Three enucleated eyes were treated with the test item, two enucleated eyes were treated as control.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no
- Time after start of exposure: not applicable

OBSERVATION TIME POINTS
- corneal opacity and corneal epithelium condition: 60, 120, 180 and 240 min
- fluorescein uptake: 240 min
- corneal swelling: 60, 120 and 240 min

SCORING SYSTEM:
- Method for Evaluation of Ocular Irritation by Slit-Lamp Biomicroscopic Examination (McDonald - Shadduck Score System)

- The scoring scheme measures the severity of corneal cloudiness and the area of the cornea involved. Severity of corneal cloudiness is graded as follows:
0 = Normal cornea. Appears with the slit-lamp as having a bright grey line on the epithelial surface and a bright grey appearance of the stroma.
1 = Some loss of transparency. Only the anterior half of the stroma is involved as observed with an optical section of the slit-lamp. The underlying structures are clearly visible with diffuse illumination, although some cloudiness can be readily apparent with diffuse illumination.
2 = Moderate loss of transparency. In addition to involving the anterior stroma, the cloudiness extends all the way to the endothelium. The stroma has lost its marble-like appearance and is homogeneously white. With diffuse, illumination, underlying structures are clearly visible.
3 = Involvement of the entire thickness of the stroma. With optical section, the endothelial surface is still visible. However, with diffuse illumination the underlying structures are just visible.
4 = Involvement of the entire thickness of the stroma. With the optical section cannot clearly visualise the endothelium. With diffuse illumination, the underlying structures cannot be seen.

The surface of the cornea relative to the area of cloudiness is divided into five grades from 0 to 4:
0 = Normal cornea with no area of cloudiness
1 = 1 to 25% area of stromal cloudiness
2 = 26 to 50% area of stromal cloudiness
3 = 51 to 75% area of stromal cloudiness
4 = 76 to 100% area of stromal cloudiness

FLUORESCEIN
- The use of fluorescein is a valuable aid in defining epithelial damage for fluorescein staining. The area can be judged as a 0 to 4 scale using the same terminology as for corneal cloudiness.

The intensity of fluorescein staining can be divided into a 0 to 4 scale:
0 = Absence of fluorescein staining
1 = Slight fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
2 = Moderate fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail.
3 = Marked fluorescein staining. Staining may involve a larger portion of the cornea. With diffuse illumination underlying structures are barely visible but are not completely obliterated.
4 = Extreme fluorescein staining. With diffuse illumination the underlying structures cannot be seen.

REFERENCE
Hackett R B and McDonald T O, Eye Irritation. In: Advances in Modern Toxicology: Dermatoxicology. 4th ed. (F Marzulli and H Maibach, eds) Hemisphere Publishing Corporation, Washington DC, 1991, pp 749-815

TOOL USED TO ASSESS SCORE:
- hand-slit lamp / biomicroscope / fluorescein

Irritation parameter:

cornea opacity score

Run / experiment:

mean after 60, 120, 180 and 240 min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

no indication of irritation

Remarks:

test item values were all 0

Irritation parameter:

fluorescein retention score

Run / experiment:

mean after 240 min

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other: test item values were all 0

Irritation parameter:

percent corneal swelling

Run / experiment:

mean after 60 min

Value:

11.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Irritation parameter:

percent corneal swelling

Run / experiment:

mean after 120 min

Value:

12.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Irritation parameter:

percent corneal swelling

Run / experiment:

mean after 240 min

Value:

13.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Other effects / acceptance of results:

negative control:
- corneal opacity: 0 (mean of 2 eyes)
- fluorescein uptake: 0 (mean of 2 eyes)
- percent corneal swelling: 8.4 at 60 min post dosing, 8.9 at 120 min post dosing, 9.6 at 240 min post dosing
- corneal epithelium condition: normal (2 eyes)

Corneal epithelium contion was normal at all timepoints for all three eyes.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was considered unlikely to have the potential to cause severe ocular irritancy in vivo.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion:

Sanders (2004) investigated acute dermal irritation of T001202 in New Zealand White rabbits (3 rabbits 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 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 T001202 (20% w/v 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 translucent after 240 minutes treatment. A mean in vitro irritancy score of 89.2 (58.9 to 111.5) was calculated after 240 minutes of treatment. Since the test item induced an IVIS ≥ 55, it is concluded that T001202 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 T001202. Three enucleated eyes of New Zealand White rabbits were treated with 0.1 mL (approx 55 mg) of test item. 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 T001202. 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), T001202 should be classified for Eye damage category 1.