Heptanal, oligomeric reaction products with aniline

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 March 2016 to 11 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study was conducted according to OECD test guidelines and in compliance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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

1

Reference substance name:

Heptanal, oligomeric reaction products with aniline

EC Number:

500-007-3

EC Name:

Heptanal, oligomeric reaction products with aniline

Cas Number:

9003-50-3

Molecular formula:

(C7H14O)n.(C6H7N)n

IUPAC Name:

Heptanal, oligomeric reaction products with aniline

Test material form:

liquid

Details on test material:

Test item: 207368/A
Identification: Hepteen Base®
Appearance: Clear amber liquid (determined by Charles River Den Bosch)
Batch: LT5C30Y170
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2016 (retest date)

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter but within 4 hours.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

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 but within 4 hours.

Transport: Eyes were collected and transported in physiological saline under cooled conditions (in a cooling box with iced elements).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

yes, concurrent negative control

Amount / concentration applied:

750 μl

Duration of treatment / exposure:

10 ± 1 minutes

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes

Number of animals or in vitro replicates:

3 corneas per test group (test substance, positive control, negative control)

Details on study design:

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) Foetal 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 ± 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 (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.

Treatment of corneas and opacity measurements
In the first test an inappropriate response of the negative control was observed (not reported), therefore a repeat test was performed. The medium from the anterior compartment was removed and 750 μl of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

Opacity measurement
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.
The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity = (((I0/I)-0.9894)/ 0.0251)
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
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 treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control 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 (Sigma-Aldrich, Germany) 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 4 mg
Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. 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 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.

Results and discussion

In vitro

Results

Irritation parameter:

other: Mean tissue viability (%)

Value:

80

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

No other effects noted.

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 (Ethanol) was 35 and was within two standard deviations of the current historical positive control mean. Therefore the test system functioned properly

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity1	Mean Permeability1	Mean In vitro Irritation Score1, 2
Negative control	-1.2	0.002	-1.1
Positive control (Ethanol)	19.7	1.039	35.3
Hepteen Base®	1.1	0.004	1.2

 

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

2In vitroirritancy score (IVIS) = mean opacity value + (15 x mean OD490value).

 

INDIVIDUAL OPACITY, PERMEABILITY AND IN VITRO SCORES

Opacity score

Treatment	Opacity before treatment	Opacity after treatment	Final opacity^1	Negative control corrected Final Opacity^2	Mean final opacity
Negative control	2.9	0.8	2.1		-1.2
	4.9	4.7	-0.1
	3.9	2.7	-1.2
Positive control	3.7	22.4	20.6	20.6	19.7
	3.5	24.0	20.4	20.4
	4.2	22.3	18.1	18.1
Hepteen base ®	5.0	5.4	0.4	0.4	1.1
	4.1	5.7	1.6	1.6
	4.2	5.5	1.2	1.2

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

2Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control.3

3In case the mean final opacity of the negative control is below zero, no correction will be made.

4Calculations are made without rounding off.

Permeability score individual values (uncorrected)

 

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control	1	0.001	0.002	0.002	0.002	0.002	0.002
	1	0.002	0.002	0.002	0.002	0.002
	1	0.004	0.003	0.003	0.003	0.003
Positive control	1	0.880	0.873	0.873	0.880	0.880
	1	0.875	0.871	0.871	0.873	0.873
	1	1.380	1.371	1.371	1.373	1.373
Hepteen base ®	1	0.009	0.008	0.008	0.008	0.008
	1	0.003	0.004	0.004	0.003	0.003
	1	0.007	0.008	0.008	0.008	0.008

 

1Calculations are made without rounding off.

Permeability score individual values (corrected)

Treatment	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD490
Positive control	1	0.878	0.84	0.871	0.877	0.877	1.039
	1	0.873	0.870	0.869	0.870	0.870
	1	1.378	0.366	1.369	1.371	1.371
Hepteen Base®	1	0.007	0.006	0.006	0.006	0.006	0.004
	1	0.001	0.001	0.002	0.001	0.001
	1	0.005	0.001	0.006	0.005	0.005

1OD490values corrected for the mean final negative control permeability (0.002).

2Calculations are made without rounding off.

In Vitroirritancy score

Treatment	Final Opacity^2	Final OD490^2	In vitro irritancy score^1
Negative control	-2.1	0.002	-2.1
	-0.1	0.002	-0.1
	-1.2	0.003	-1.2
Positive control	20.6	0.877	33.8
	20.4	0.870	33.5
	18.1	1.371	38.7
Hepteen base ®	0.4	0.006	0.5
	1.6	0.001	1.6
	1.2	0.005	1.3

1In vitroirritancy score (IVIS) = opacity value + (15 x OD490value).

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

Historical control data for the BCOP studies

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.9-3.0	-0.016-0.019	-2.8-3.0	35.9-63.9
Mean	0.22	0.00	0.30	50.74
SD	1.12	0.01	1.18	10.34The 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 meanin vitroirritancy score of the positive control (Ethanol) was 35 and was 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. Hepteen Base® did not induce ocular irritation through both endpoints, resulting in a meanin vitroirritancy score of 1.2 after 10 minutes of treatment. Since Hepteen Base® induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
n	39	34	36	12

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 February 2015 to January 2016.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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 (Ethanol) was 35 and was 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.

Hepteen Base® did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.2 after 10 minutes of treatment.

Hepteen Base® induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The eye damage of Hepteen Base® was tested through topical application on bovine eyes for 10 minutes.The study procedures were based on the most recent OECD guideline.

 

Batch LT5C30Y170 of Hepteen Base® was a clear amber liquid a purity of 97.3%. The test item was applied as it is (750 μl) directly 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 (Ethanol) was 35 and was 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.

 

Hepteen Base® did not induce ocular irritation through both endpoints, resulting in a mean in vitroirritancy score of 1.2 after 10 minutes of treatment.

Since Hepteen Base® induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.