Reaction mass of (Z)-1-chloro-1-propene and 2-chloropropane and 2-chloropropene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD guideline 437

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline for testing of chemicals-guideline 437: "Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants"

Deviations:

no

Principles of method if other than guideline:

The Bovine Corneal Opacity and Permeability Assay (BCOP) measures two important components which are predictive of irritation, corneal opacity and permeability.
The test consists of topical application of Reaction mass containing mainly 2-chloropropene on the epithelium of the bovine cornea for 10 minutes at room temperature and at 32°C. Reaction mass containing mainly 2-chloropropene is applied undiluted. After exposure the cornea is thoroughly rinsed to remove the test substance and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-chloropropene

EC Number:

209-187-5

EC Name:

2-chloropropene

Cas Number:

557-98-2

Molecular formula:

C3H5Cl

IUPAC Name:

2-chloroprop-1-ene

Constituent 2

Reference substance name:

2-chloropropane

EC Number:

200-858-8

EC Name:

2-chloropropane

Cas Number:

75-29-6

Molecular formula:

C3H7Cl

IUPAC Name:

2-chloropropane

Constituent 3

Reference substance name:

1-chloropropene

EC Number:

209-675-8

EC Name:

1-chloropropene

Cas Number:

590-21-6

IUPAC Name:

1-chloroprop-1-ene

Details on test material:

Identification Reaction mass containing mainly 2-chloropropene
Molecular formula C3H5Cl
Molecular weight 76.53
CAS Number 557-98-2
Description Clear light yellow to brown liquid (determined at NOTOX)
Batch RBA100301A
Purity Minimum 60.0%
Test substance storage In refrigerator (2-8°C) in the dark
Stability under storage conditions Stable
Expiry date 08 March 2011 (allocated by NOTOX, 1 year after receipt of the test substance)

Test animals / tissue source

Species:

other: Bovine cornea

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter on the same day.

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.

Transport: Eyes were collected and transported in physiological saline in a suitable container.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

750 µl of the negative and positive control substances and test substance were introduced onto the epithelium of the cornea.

Duration of treatment / exposure:

10 +/- 1 minutes

Observation period (in vivo):

120 +/- 10 minutes

Number of animals or in vitro replicates:

3 bovine cornea per conditions were used (Negative control-Positive control-Test substance)
Since Reaction mass containing mainly 2-chloropropene has a very low boiling point (23°C), additional corneas were treated at room temperature, to compare the results with the treatment at 32°C. Three additional corneas were treated with 750 µl test substance and incubated for
10 +/- 1 minutes at room temperature.

Details on study design:

Preparation of corneas
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation were discarded.
The isolated corneas were stored at 32 +/- 1°C in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v)
L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, 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, 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.

Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 µl of the negative and positive control substances and test substance were 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 substance over the entire cornea. Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1°C. After the incubation the control or test substance was removed and the epithelium was washed at least three times with cMEM. 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.
Since Reaction mass containing mainly 2-chloropropene has a very low boiling point (23°C), additional corneas were treated at room temperature, to compare the results with the treatment at 32°C. Three additional corneas were treated with 750 µl test substance and incubated for
10 +/- 1 minutes at room temperature. After that the handling was as described above.

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 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.

Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck, Darmstadt, 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/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 (Multiskan spectrum, Thermo labsystems, Breda, The Netherlands). 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.

Electronic data capture
Observations/measurements in the study will be recorded electronically using the following programme: Multiskan spectrum version 1.00 (Thermo labsystems, Breda, The Netherlands) for optical density measurement.

Results and discussion

In vivo

Results

Irritation parameter:

overall irritation score

Remarks:

Opacity measurement

Basis:

mean

Time point:

other: 10 minutes

Score:

<= 55.1

Remarks on result:

other: Not severely irritant

Irritant / corrosive response data:

Reaction mass containing mainly 2-chloropropene was tested neat at room temperature and at 32 °C.

Table 1 summarizes the opacity, permeability and in vitro irritancy scores of Reaction mass containing mainly 2-chloropropene and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Appendix I, Table 2-5.

The individual in vitro irritancy scores for the negative controls ranged from 0 to 0.1. The individual positive control in vitro irritancy scores ranged from 107 to 137 for Benzalkonium Chloride (Appendix I, Table 5). The corneas treated with the positive control substances were turbid after the 10 minutes of treatment.

The corneas treated with Reaction mass containing mainly 2-chloropropene showed opacity values ranging from 6 to 8 at room temperature and from 8 to 9 at 32°C. The permeability values ranged from 1.536 to 3.060 at room temperature and from 2.634 to 2.862 at 32°C. The corneas were slightly turbid after the 10 minutes of treatment with Reaction mass containing mainly 2-chloropropene at both temperatures. The test substance had evaporated from the corneas treated at 32°C after the 10 minutes treatment period. Hence, the in vitro irritancy scores ranged from 31 to 53 at room temperature and from 48 to 52 at 32°C after 10 minutes of treatment with Reaction mass containing mainly 2-chloropropene.

Any other information on results incl. tables

Table1           Summary of opacity, permeability andin vitroscores

 

Treatment	Mean Opacity^1	Mean Permeability^1	MeanIn vitroIrritation Score^1, 2
Negative control	0	0.000	0
Positive control (Benzalkonium Chloride)	78	3.232	127
Reaction mass containing mainly 2-chloropropene^3	7	2.516	45
Reaction mass containing mainly 2-chloropropene^4	9	2.762	50

 

1          Calculated using the negative control mean opacity and mean permeability values.

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

3           Reaction mass containing mainly 2-chloropropene was tested at room temperature

4           Reaction mass containing mainly 2-chloropropene was tested at 32°C

APPENDIXI    INDIVIDUAL OPACITY, PERMEABILITY AND IN VITRO SCORES

 

Table2           Opacity score

Eye	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity	Mean Opacity
Negative control
1	0	1	1	0		0
2	0	1	1	0
3	0	1	1	0
Positive control
4	0	85	85	84		78
5	0	73	73	72
6	0	80	80	79
Reaction mass containing mainly 2-chloropropene at room temperature
16	0	8	8	7		7
19	0	7	7	6
18	0	9	9	8
Reaction mass containing mainly 2-chloropropene at 32°C
13	0	10	10	9		9
14	0	10	10	9
15	0	9	9	8

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

 

Table3           Permeability scoreindividual values (uncorrected)

Eye	Dilution factor	OD490 1	OD490 2		OD490 3		Average OD		Final OD		Mean final negative control
Negative control
1	1	0.012		0.009		0.014		0.012		0.012		0.014
2	1	0.013		0.012		0.012		0.012		0.012
3	1	0.022		0.017		0.018		0.019		0.019
Positive control(Benzalkonium Chloride)
4	6	0.616		0.602		0.602		0.607		3.642
5	6	0.411		0.406		0.400		0.406		2.436
6	6	0.636		0.655		0.645		0.645		3.870
Reaction mass containing mainly 2-chloropropeneat room temperature
16	6	0.533		0.522		0.518		0.524		3.144
19	6	0.512		0.508		0.497		0.506		3.036
18	6	0.275		0.269		0.265		0.270		1.620
Reaction mass containing mainly 2-chloropropeneat 32°C
13	6	0.498		0.491		0.483		0.491		2.946
14	6	0.480		0.476		0.480		0.479		2.874
15	6	0.457		0.448		0.455		0.453		2.718

Table4           Permeability score individual values (corrected)

Eye	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 OD
Negative control
1	1	-0.002	-0.005	0.000	-0.002	-0.002	0.000
2	1	-0.001	-0.002	-0.002	-0.002	-0.002
3	1	0.008	0.003	0.004	0.005	0.005
Positive control(Benzalkonium Chloride)
4	6	0.602	0.588	0.588	0.593	3.558	3.232
5	6	0.397	0.392	0.386	0.392	2.352
6	6	0.622	0.641	0.631	0.631	3.786
Reaction mass containing mainly 2-chloropropeneat room temperature
16	1	0.519	0.508	0.504	0.510	3.060	2.516
19	1	0.498	0.494	0.483	0.492	2.952
18	1	0.261	0.255	0.251	0.256	1.536
Reaction mass containing mainly 2-chloropropeneat 32°C
13	1	0.484	0.477	0.469	0.477	2.862	2.762
14	1	0.466	0.462	0.466	0.465	2.790
15	1	0.443	0.434	0.441	0.439	2.634

¹       OD₄₉₀values corrected for the mean final negative control permeability (0.014).

 

Table5           In Vitro irritancy score

Eye	Negative control correctedFinal Opacity	Negative control correctedFinal OD490	In vitroIrritancy Score^1
Negative control
1	0	-0.002	0.0
2	0	-0.002	0.0
3	0	0.005	0.1
Positive control(Benzalkonium Chloride)
4	84	3.558	137.4
5	72	2.352	107.3
6	79	3.786	135.8
Reaction mass containing mainly 2-chloropropeneat room temperature
16	7	3.060	52.9
19	6	2.952	50.3
18	8	1.536	31.0
Reaction mass containing mainly 2-chloropropeneat 32°C
13	9	2.862	51.9
14	9	2.790	50.9
15	8	2.634	47.5

 

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

 

 

 

Applicant's summary and conclusion

Interpretation of results:

Category 2B (mildly irritating to eyes)

Remarks:

Migrated information Criteria used for interpretation of results: other:

Conclusions:

The negative control responses of the opacity and permeability values 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 (10% (w/v) Benzalkonium Chloride) was 127 and was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The mean in vitro irritancy scores were 45 and 50 after 10 minutes of treatment with Reaction mass containing mainly 2-chloropropene at room temperature and at 32°C, respectively. The corneas were slightly turbid after the 10 minutes of treatment. Although the test substance had evaporated from the corneas treated at 32°C after the 10 minutes treatment period, no obvious difference was observed between the corneas treated at room temperature and at 32°C.

Since the mean in vitro irritancy scores for Reaction mass containing mainly 2-chloropropene were below 55.1 after 10 minutes treatment, Reaction mass containing mainly 2-chloropropene is considered to be not severely irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Executive summary:

Screening for the eye irritancy potential of Reaction mass containing mainly 2-chloropropene using the Bovine Corneal Opacity and Permeability test (BCOP test).

 

This report describes the ocular irritation properties of Reaction mass containing mainly 2-chloropropene on an isolated bovine cornea. The possible ocular irritancy of Reaction mass containing mainly 2-chloropropene was tested through topical application for 10 ± 1 minutes. Due to the volatility of the test substance, Reaction mass containing mainly 2-chloropropene was tested at room temperature and at 32°C.

 

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

 

Batch100301A of Reaction mass containing mainly 2-chloropropene was a clear light yellow to brown liquid. The test substance was applied as it is (750 µl) directly on top of the corneas.

 

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 127 and was within the historical positive control data range.It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

The mean in vitro irritancy scores were 45 and 50 after 10 minutes of treatment with Reaction mass containing mainly 2-chloropropene at room temperature and at 32°C, respectively. The corneas were slightly turbid after the 10 minutes of treatment. Although the test substance had evaporated from the corneas treated at 32°C after the 10 minutes treatment period, no obvious difference was observed between the corneas treated at room temperature and at 32°C.

 

Since the mean in vitro irritancy scores for Reaction mass containing mainly 2-chloropropene were below 55.1 after 10 minutes treatment, Reaction mass containing mainly 2-chloropropene is considered to be not severely irritant or corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.