Benzonitrile, 4-[(1,6-dihydro-4-hydroxy-6-oxo-2-pyrimidinyl)amino]-

Endpoint summary

Description of key information

Skin irritation:

Skin irritation potential was assessed with a K2 in vivo skin irritation study performed according to the OECD guideline N°404 and the EU Method B.4 in New Zealand White rabbits. No evidence for skin irritation was noted for T002326.

Eye irritation:

In a K1 Bovine Corneal Opacity and Permeability (BCOP) test, performed according to OECD guideline 437 and EU method B.47, T002326 did not induced occular irritation (Eurlings, 2015). 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

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-05-28 to 2003-05-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed according 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Remarks:

: No information on the test substance was provided (including purity information); limited information on the preparation of the test substance and test site; limited information on the application of the test substance and the test animals

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

: No information on the test substance was provided (including purity information); limited information on the preparation of the test substance and test site; limited information on the application of the test substance and the test animals

GLP compliance:

no

Remarks:

This study was conducted in a facility operating to Good Laboratory Practice within the UK national GLP monitoring program, but 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 test system and environmental conditions:

TEST ANIMALS: no data

ENVIRONMENTAL CONDITIONS: no data


IN-LIFE DATES: From: 2003-05-28 To: 2003-05-31

Type of coverage:

semiocclusive

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

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:

4 hours

Observation period:

Skin reactions were recorded 1, 24, 48 and 72 hours after administration.

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: no data
- % coverage: no data
- Type of wrap if used: no data

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no data
- Time after start of exposure: 4 hours

SCORING SYSTEM:
- Skin irritation/corrosion was scored according to the Draize system.

Irritation parameter:

primary dermal irritation index (PDII)

Basis:

mean

Time point:

other: all time points

Score:

0

Max. score:

8

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animals number 69, 70 and 71

Time point:

other: all time points

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Irritation parameter:

edema score

Basis:

mean

Remarks:

animals number 69, 70 and 71

Time point:

other: all time points

Score:

0

Max. score:

4

Reversibility:

other: not applicable - score 0

Irritant / corrosive response data:

- No evidence of skin irritation was noticed at any time at any test site.

Interpretation of results:

GHS criteria not met

Conclusions:

The test material did not meet the criteria for classification as irritant or corrosive according to EU labelling regulations Commission Directive 2001/59/EC. No symbol and risk phrase are required.

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 2015-06-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well documented GLP study according to OECD guideline 437 and EU method B.47

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

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: I15BB0824
- Expiration date of the lot/batch: 2015-08-28 (retest date)
- Purity test date: 2015-03-17

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: not applicable
- 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: none

FORM AS APPLIED IN THE TEST : slight beige powder

OTHER SPECIFICS:

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:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 400 mg, the test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered

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:

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

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:

TREATMENT METHOD
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 (approximately 400 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°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.

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:

Test item after 240 min treatment

Value:

-1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: -1.7 to -0.3

Irritation parameter:

cornea opacity score

Run / experiment:

Test item after 240 min treatment

Value:

-2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: -2.7 to -0.7

Irritation parameter:

other: permeability score

Run / experiment:

Test item after 240 min treatment

Value:

0.058

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Range: 0.028 to 0.085

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: 0.0 (-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 (-2.7 to -0.7)

OTHER EFFECTS:
The corneas treated with the positive control were turbid after the 240 minutes of treatment.
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.

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.
- It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.1 (-1.7 to -0.3) after 240 minutes of treatment.
Since the test item 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)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

Sanders (2004) investigated acute dermal irritation of T002326 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.

There were no signs of irritation for any animal during the 72-hour observation period the following the 4-hour exposure period. The test item did not meet the criteria for classification as irritant or corrosive according to EU labelling regulations Commission Directive 2001/59/EC.

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. Approximatively 400 mg of the test item was added pure on the top of 3 corneas during 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 T002326 were clear after 240 minutes of treatment. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.1 (-1.7 to -0.3). Since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

In addition, a rabbit enucleated eye test (REET) was performed by Sanders (2004) to assess the ocuar irritancy potential of T002326.

Three enucleated eyes, obtained from the New Zealand White strain of rabbit, were treated with 0.1 ml of T002326. 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.

The test item had no effect on corneal opacity or corneal epithelium condition at any time, although it did adhere to the corneas. There was no fluorescein uptake by any eye at any time observed, and only minimal corneal swelling (2.0-3.2%). Based on the obtained results, the test item was not considered to have the potential to cause severe occular irritancy in vivo.

Justification for classification or non-classification

Skin irritation:

According to the in vivo acute dermal irritation study 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), no classification is required for eye irritation or serious eye damage.