4-chloroformylphthalic anhydride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 May 2015-12 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 437. Furthermore, functional model conditions and references to historical control data are included in the report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

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:

Adopted July 26, 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Amended by EC No. 1153/2010 No. L142, 09 December 2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The Ocular Toxicity Working Group (OTWG) of the ICCVAM and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): BCOP Test Method, March 2006

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: 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.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-chloroformylphthalic anhydride

EC Number:

214-874-8

EC Name:

4-chloroformylphthalic anhydride

Cas Number:

1204-28-0

Molecular formula:

C9H3ClO4

IUPAC Name:

1,3-dioxo-1,3-dihydro-2-benzofuran-5-carbonyl chloride

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): Trimellitic anhydride chloride
For more details see Confidential details on test material

Specific details on test material used for the study:

4-chloroformylphthalic anhydride

Test animals / tissue source

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 (Vitelco,’s Hertogenbosch, The Netherlands)
- Number of animals: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): Not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Instructions were given to avoid damaging the corneas during excision. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: as soon as possible after slaughter

Test system

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): 304.5 to 325.1 mg of test item was directly applied on each cornea (completely covered).
- Concentration (if solution): Not applicable

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

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): physiological saline

Duration of treatment / exposure:

4 hours ± 10 minutes at 32 ± 1 °C in a horizontal position

Duration of post- treatment incubation (in vitro):

None

Number of animals or in vitro replicates:

Total: 9 corneas - 3 corneas/group for test item, negative and positive controls

Details on study design:

SELECTION AND 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.

QUALITY CHECK OF THE ISOLATED CORNEAS : 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 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES : 3 corneas/group

NEGATIVE CONTROL USED : physiological saline

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

APPLICATION DOSE AND EXPOSURE TIME : The anterior compartment received the test item (304.5 to 325.1 mg) or negative or positive control at a volume (750 μL) on the surface of the corneae. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas. The corneae were incubated in a horizontal position for 4 hours ± 5 minutes at 32 ± 1 °C.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: Yes.
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.

REMOVAL OF TEST SUBSTANCE:
- Number of washing steps after exposure period: 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).

- POST-EXPOSURE INCUBATION: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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 substance 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 substance 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.
- Corneal permeability: 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.
- Others (e.g, pertinent visual observations, histopathology): Possible pH effects of the test substance on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA:
In vitro scrore range: UN GHS:
- =< 3 - No category
- > 3; =< 55 - No prediction can be made
- > 55 - Category 1

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean/3 corneas incubated 2 hours

Value:

42.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0

Positive controls validity:

valid

Remarks:

144.0

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas were translucent with spots after the 240 minutes of treatment with Trimellitic anhydride chloride. A pH effect of the test substance was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline:
The positive control should elicit an In Vitro Irritancy Score that falls within two standard deviations of the historical mean (2 x SD 18.75 to 95.22) for the laboratory.
The negative control mean opacity change value should be ≤2.0 and the permeability mean value ≤0.050 (assays performed from April 2012 to April 2015).

Any other information on results incl. tables

Table 1 - Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity^1	Mean Permeability^1	Mean In Vitro Score^1, 2
Negative control	0.0	0.000	0.0
Positive control (20% (w/v) imidazole)	84.3	3.975	144.0
Test item	42.3	-0.017	42.1

¹Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

²In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Applicant's summary and conclusion

Interpretation of results:

other: no prediction can be made

Conclusions:

Trimellitic anhydride chloride induced ocular irritation through only one endpoint (opacity), resulting in a mean in vitro irritancy score of 42.1 after 240 minutes of treatment.
With an IVIS at 42.1, no prediction can be made as the result is outside the decision criteria (i.e. >3 or =<55)

Executive summary:

The eye hazard potential of the Trimellitic anhydride chloride was evaluated using the Bovine Corneal Opacity and Permeability test (BCOP test), under GLP compliance, according to the OECD Guideline No. 437.

The corneal damage potential of test substance was assessed using fresh bovine cornea. 304.5 to 325.1 mg of test item was applied topically on cornea for 240 minutes at 32 ± 1 °C. After incubation, the solutions and the test compound were removed and the epithelium was washed. Each cornea was inspected visually and then, corneal opacity was measured. Three corneas were used for each treated series (test item; negative control: physiological saline; positive control: 20% (w/v) imidazole). Following the opacity measurement, the permeability of the corneas to sodium fluorescein was evaluated. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score.

 

The individual in vitro irritancy scores for the negative controls ranged from -0.3 to 0.6. The individual positive control in vitro irritancy scores ranged from 120.9 to 153.6. The corneas treated with the positive control item 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 (2.0 and 0.050, respectively) indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control was 144.0 and within two standard deviations of the current historical positive control mean (68 - 135). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The corneas treated with Trimellitic anhydride chloride were translucent with spots after the 240 minutes of treatment. A pH effect of the test substance was observed on the rinsing medium. Hence, Trimellitic anhydride chloride induced ocular irritation through only one endpoint (opacity), resulting in a mean in vitro irritancy score of 42.1 after 240 minutes of treatment (in vitro irritancy scores ranged from 31.4 to 49.4) and, the mean IVIS was greater than 3 and lower than 55 (i.e. 42.1).

 

Therefore, no prediction on the classification can be made for eye irritation or serious eye damage, according to the GHS Regulation.