Terephthalaldehyde

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

BCOP assay OECD 437

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Thie study will be available on 27/07/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Terephthalaldehyde

EC Number:

210-784-8

EC Name:

Terephthalaldehyde

Cas Number:

623-27-8

Molecular formula:

C8H6O2

IUPAC Name:

benzene-1,4-dicarbaldehyde

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Batch (Lot) Number: 180401
Expiry date: 01 April 2020 (retest date)
Physical Description: Light yellow powder
Purity/Composition: 99.6%
Storage Conditions: At room temperature

Test animals / tissue source

Species:

cattle

Strain:

not specified

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Since no workable suspension of Terephthaldehyde in physiological saline could be obtained,
the test item was used as delivered by the sponsor and added pure on top of the corneas. Terephthaldehyde was weighed in a bottle and applied directly on the corneas in
such a way that the cornea was completely covered (306.3 to 329.6 mg)

Duration of treatment / exposure:

Corneas were incubated in a horizontal
position for 240 +-10 minutes at 32 +- 1C.

Duration of post- treatment incubation (in vitro):

N/A

Number of animals or in vitro replicates:

3 (three)

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 (Earle’s Minimum Essential
Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine
(Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated
corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen,
Germany) 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 +- 1C. The corneas were
incubated for the minimum of 1 hour at 32 +- 1C.
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 (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea
was read against a cMEM 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 (three)
NEGATIVE CONTROL USED
750 l of the negative control physiological saline
SOLVENT CONTROL USED (if applicable)
N/A
POSITIVE CONTROL USED
20% (w/v) Imidazole solution (positive control)
APPLICATION DOSE AND EXPOSURE TIME
Terephthaldehyde was weighed in a bottle and applied directly on the corneas in
such a way that the cornea was completely covered (306.3 to 329.6 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 +- 1C.

TREATMENT METHOD: [closed chamber / open chamber]
not specified
POST-INCUBATION PERIOD: yes/no. If YES please specify duration
No
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 (Earle’s Minimum Essential Medium Life Technologies).
- POST-EXPOSURE INCUBATION:
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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
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 l 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 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.
- Others (e.g, pertinent visual observations, histopathology): (please specify)
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.
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

yes

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

ca. 20

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

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.
All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.

Any other information on results incl. tables

Terephthaldehyde was tested as it is.

Table 1 of Appendix 1 summarizes the opacity, permeability and in vitro irritancy scores of

Terephthaldehyde and the controls.  The opacity, permeability and in vitro scores of the

individual corneas are shown in Appendix 2, Table 2 - 5.  

The individual in vitro irritancy scores for the negative controls ranged from 0.9 to 2.0.  The

corneas treated with the negative control item were clear after the 240 minutes of treatment.

The individual positive control in vitro irritancy scores ranged from 137 to 173 (Appendix 2,

Table 5).  The corneas treated with the positive control were turbid after the 240 minutes of

treatment.

The corneas treated with Terephthaldehyde showed opacity values ranging from 7.4 to 14 and

permeability values ranging from 0.023 to 1.120.  The corneas were translucent after the

240 minutes of treatment with Terephthaldehyde.  No pH effect of the test item was observed

on the rinsing medium.  Hence, the in vitro irritancy scores ranged from 7.7 to 27 after

240 minutes of treatment with Terephthaldehyde.

 

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 159 and within two standard deviations of the current historical positive control mean

(Appendix 3, Table 6).  It was therefore concluded that the test conditions were adequate and

that the test system functioned properly.  

Terephthaldehyde induced ocular irritation through both endpoints, resulting in a mean in

vitro irritancy score of 20 after 240 minutes of treatment.

Table1          
Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	1.5	0.006	1.6
Positive control	123	2.387	159
Terephthaldehyde	11	0.659	20

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

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

Table2          
Opacity Score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	2.2	4.1	1.9			1.5
	3.5	4.3	0.8
	2.7	4.6	1.9
Positive control	3.3	130.6	127.4	126		123
	3.4	145.1	141.7	140
	2.3	107.5	105.2	104
Test item	3.7	18.9	15.3	14		11
	3.1	15.2	12.2	11
	1.8	10.7	8.9	7.4

Calculations are made without rounding off.

¹  Final Opacity = Opacity after treatment – Opacity before treatment.

²  Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

 

Table3          
Permeability ScoreIndividual Values (Uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2		OD490 3		Average OD		Final OD		Mean final negative control
Negative control	1	0.003		0.002		0.003		0.003		0.003		0.006
	1	0.008		0.008		0.008		0.008		0.008
	1	0.007		0.008		0.007		0.007		0.007
Positive control	6	0.505		0.471		0.428		0.468		2.808
	6	0.433		0.348		0.320		0.367		2.202
	6	0.414		0.365		0.351		0.377		2.260
Test item	1	0.837		0.843		0.839		0.840		0.840
	1	1.145		1.127		1.107		1.126		1.126
	1	0.029		0.029		0.029		0.029		0.029

Calculations are made without rounding off.

 

Table4          
Permeability Score Individual Values (Corrected)

Treatment	Dilution factor	Negative control corrected OD490 1^1	Negative control corrected OD490 2^1	Negative control corrected OD490 3^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	6	0.499	0.465	0.422	0.462	2.772	2.387
	6	0.427	0.342	0.314	0.361	2.166
	6	0.408	0.359	0.345	0.371	2.224
Test item	1	0.831	0.837	0.833	0.834	0.834	0.659
	1	1.139	1.121	1.101	1.120	1.120
	1	0.023	0.023	0.023	0.023	0.023

Calculations are made without rounding off.

¹  OD490 values corrected for the mean final negative control permeability (0.006).

 

 

Table5          
In VitroIrritancy Score

Treatment		Final Opacity^2		Final OD490^2		In vitroIrritancy Score^1
Negative control		1.9		0.003		2.0
		0.8		0.008		0.9
		1.9		0.007		2.0
Positive control		126		2.772		167
		140		2.166		173
		104		2.224		137
Terephthaldehyde		14		0.834		26
		11		1.120		27
		7.4		0.023		7.7

¹  In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

²  Positive control and test item are corrected for the negative control.

  Table6          
Historical Control Data for the BCOP Studies

	Negative control			Positive control
	Opacity	Permeability	In vitroIrritancy Score	In vitroIrritancy Score
Range	-5.4 – 5.2	-0.011 - 0.205	-5.3 – 5.4	86.5 – 211.4
Mean	0.55	0.02	0.78	138.42
SD	1.84	0.02	1.90	26.57
n	166	166	166	166

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of May 2015 to May 2018.

 

Applicant's summary and conclusion

Interpretation of results:

other: further testing required as per top down testing strategy

Conclusions:

In conclusion, since Terephthaldehyde induced an IVIS > 3 ≤ 55, no prediction on the
classification can be made.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Terephthaldehyde as

measured by its ability to induce opacity and increase permeability in an isolated bovine

cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated

bovine corneas.  The eye damage of Terephthaldehyde was tested through topical application

for approximately 240 minutes.  

The study procedures described in this report were based on the most recent OECD guideline.

Batch 180401 of Terephthaldehyde was a light yellow powder.  Since no workable

suspension in physiological saline could be obtained, the test item was used as delivered and

added pure on top of the corneas.

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 159 and within two standard deviations of the current historical positive control mean.  It

was therefore concluded that the test conditions were adequate and that the test system

functioned properly.  

Terephthaldehyde induced ocular irritation through both endpoints, resulting in a mean in

vitro irritancy score of 20 after 4 hours of treatment.

In conclusion, since Terephthaldehyde induced an IVIS > 3 ≤ 55, no prediction on the

classification can be made.