Fatty acids, C9-13-neo-, barium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-14

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)

Version / remarks:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C9-13-neo-, barium salts

EC Number:

295-361-6

EC Name:

Fatty acids, C9-13-neo-, barium salts

Cas Number:

92044-82-1

IUPAC Name:

Fatty acids, C9-13-neo-, barium salts

Test material form:

solid

Details on test material:

- State of aggregation: white solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in a sealed container

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was crushed to a fine powder using a mortar and a pestle.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories
- Time interval prior to initiating testing: immediately after arrival of the eyes, cornea preparation was initiated and was used for BCOP testing on the same day.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): about 750 mg of the test item
Since it was not possible to get a solution with the test item, it was administered directly and moistened with a drop of physiological saline (0.9% NaCl).

Duration of treatment / exposure:

4 hours

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not required

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- eyes were examined for defects and any defective eyes were discarded. Eyes with scratches or any kind of opacity were not used.
- tissue surrounding the eyeball was pulled away and the cornea was excised.
- isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with pre-warmed MEM (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete MEM).
- corneas were incubated for one hour at 32 ± 1 °C for equilibration in an air incubator.

QUALITY CHECK OF THE ISOLATED CORNEAS
- after the equilibration period, the medium was removed from both chambers and replaced with fresh complete MEM.
- an initial measurement was performed on each of the corneas using the opacitometer.
- three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas.
- the illuminance of each cornea was read and recorded.
- only corneas that had an initial illuminance reading I > I0/1.1651 lux (an equivalent to the opacity threshold of 7 as listed in OECD 437) were used for the assay.

APPLICATION DOSE AND EXPOSURE TIME
- medium was removed from the anterior chamber and replaced with the test item or control.
- 750 µL of the control substance (closed-chamber method) or enough test item to completely cover the cornea (about 750 mg, open-chamber method) were introduced into the anterior chamber.
- after 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed.

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- epithelium was washed at least three times with MEM (containing phenol red).
- once the medium was free of test substance, the cornea was finally rinsed with complete MEM (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- anterior chamber was refilled with complete MEM and an illuminance measurement was performed.
- corneas were visually examined for tissue peeling, residual test chemical and non-uniform opacity patterns and observation were recorded.
- after the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- posterior chamber was refilled with fresh complete MEM.
- 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C in horizontal position.
- then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Evaluation of the opacity:
- the following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity = ((I0/I) - b)/ a
with a = 0.025 and b = 0.9894
- value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically. This I0 value was than calculated to the respective data of the opacitometer and the data according to guideline (opacity < 7). So the initial illuminance could be calculated and corneas below this value were discarded.
- change in opacity for each cornea (test item, positive and negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values of test item treated cornea or positive control were corrected by subtracting from each the average change in opacity observed for the negative-control corneas to obtain a corrected opacity. The mean corrected opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

Evaluation of the permeability:
- mean OD490 for the blank cuvettes was calculated.
- mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490).
- any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor.
- final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
- mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
To determine the IVIS of the positive control and the test item, the corrected opacity and OD490 values are used.

ACCEPTABILITY CRITERIA
- the BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Run / experiment:

Experiment 1

Value:

293.43

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

not valid

Remarks on result:

other: The in vitro irritation score obtained with the positive control did not fall within the two standard deviations of the current historical mean. Therefore, the assay was not considered to be valid and the experiment was repeated (Experiment 2).

Results 2

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Run / experiment:

Experiment 2

Value:

237.92

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

EXPERIMENT 1:

-Measurement after treatment:
All 3 corneas treated with Fatty acids, C9-C13-neo, barium salts showed severe opacity of the tissue and a slight increase of permeability.
- Acceptance of results:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
- Acceptance criteria met for positive control: the in vitro irritation score obtained with the positive control did not fall within the two standard deviations of the current historical mean. Therefore, the assy was not considered to be valid and the experiment was repeated.

EXPERIMENT 2:

- Measurement after treatment:
Relative to the negative control, the test item caused a severe increase of corneal opacity and a slight increase of permeability in all 3 corneas.

- Visual Observation after treatment:
All 3 corneas treated with Fatty acids, C9-C13-neo, barium salts showed very severe opacity of the tissues.
The experiment showed residual Fatty acids, C9-C13-neo, barium salts on the corneas. The results of the opacity measurements are therefore confounded by the presence test item material. Since the residual material could not be removed by non-invasive methods, such as additional rinsing, it is concluded that the in vitro eye irritancy potential of Fatty acids, C9-C13-neo, barium salts cannot be assessed in the bovine corneal opacity and permeability assay. Due to these technical limitations, the study cannot be performed with Fatty acids, C9-C13-neo, barium salts according to the guideline OECD437.

- Acceptance of results:
The test is valid since the IVIS of the positive control falls within two standard deviations of the current historical mean and opacity and permeability of the negative control are less than the respective established upper limits for background opacity and permeability.

The evaluation of acceptability criteria was performed by using the following historical data:
- for evaluation of the validity of the positive control, the historical mean IVIS score as obtained from 2015 until 2017 was used (please refer to the tables in the field "Any other information on results incl. tables").
- for the evaluation of the validity of the negative control, the historical upper limits of the opacity and permeability values as obtained in 2017 were used (please refer to the tables in the field "Any other information on results incl. tables" below)

Please also refer for results to the field "Any other information on results incl. tables" below

Any other information on results incl. tables

EXPERIMENT 2 valid

Table 1: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	0.19	0.97	0.79
2		0.19	0.97	0.79
3		0.12	0.66	0.54
MV		0.13	0.87	0.71
4	Positive Control	1.40	99.43	98.03	97.33
5		1.43	79.29	77.86	77.15
6		1.01	105.47	104.46	103.75
MV		1.28	94.73	93.45	92.74
7	Test Item	0.39	227.17	226.78	226.08
8		-0.25	187.05	187.30	186.59
9		-0.41	212.76	213.17	212.47
MV		-0.09	209.00	209.09	208.38
MV = mean value

Table 2:  Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.006
2		0.005
3		0.005
MV		0.005
4	Positive Control	2.715	2.710
5		3.240	3.235
6		5.280	5.275
MV		3.745	3.740
7	Test Item	1.995	1.990
8		2.050	2.045
9		1.880	1.875
MV		1.975	1.970
MV = mean value

Table 3:  In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative Control	0.79	0.006
2		0.79	0.005
3		0.54	0.005
MV		0.71	0.005	0.79
4	Positive Control	97.33	2.710
5		77.15	3.235
6		103.75	5.275
MV		92.74	3.740	148.84
7	Test Item	226.08	1.990
8		186.59	2.045
9		212.47	1.875
MV		208.38	1.970	237.92
MV = mean value

Table 4: Historical mean In Vitro Irritation Score of the Positive Control (Imidazole 20%) from February 2015 until May 2018

	IVIS Positive Control - Imidazole 20 %
Mean Value (MV)	123.98
Standard Deviation (SD)	17.69
MV- 2xSD	88.60
MV+2xSD	159.37
Number of Replicates providing Historical Mean: 36

Positive controls are updated after every single experiment or at least every 3 months.

Table 5: Historical Data on Opacity and Permeability of the Positive Control (Imidazole 20%) from August 2017 until May 2018

Replicates Providing Historical Mean	Cornea No.	Opacity		Permeability		IVIS
		Change of Opacity Value	Corrected Opacity Value	OD490 Value	Corrected OD490 Value
1	4	122.785	121.861	0.662	0.624	133.420
	5	117.173	116.249	1.220	1.182
	6	102.655	101.731	2.260	2.222
2	4	108.553	106.381	1.473	1.450	123.050
	5	79.491	77.319	2.465	2.442
	6	83.618	81.446	3.065	3.042
3	4	55.644	56.308	2.200	2.189	92.540
	5	71.511	72.175	1.348	1.337
	6	65.148	65.812	2.040	2.029
4	4	68.39	67.72	2.400	2.383	112.690
	5	70.88	70.21	2.810	2.793
	6	94.23	93.56	1.945	1.928
5	4	70.53	70.35	1.296	1.284	102.440
	5	78.68	78.50	1.363	1.351
	6	91.69	91.51	1.840	1.828
6	4	95.54	94.92	1.478	1.467	114.650
	5	83.58	82.96	1.500	1.489
	6	91.11	90.49	2.095	2.084
7	4	89.35	88.86	2.855	2.838	149.420
	5	117.36	116.87	2.215	2.198
	6	130.15	129.66	2.505	2.488
8	4	80.99	80.24	2.140	2.127	120.110
	5	78.40	77.65	3.070	3.057
	6	76.27	75.51	3.290	3.277
9	4	98.03	97.33	2.715	2.710	148.838
	5	77.86	77.15	3.240	3.235
	6	104.46	103.75	5.280	5.275
Mean Value (MV)		89.040	88.390	2.251	2.234	121.906
Standard Deviation (SD)		18.814	18.573	0.916	0.919	19.360
MV- 2xSD		51.412	51.244	0.419	0.396	83.187
MV+2xSD		126.667	125.536	4.082	4.073	160.626

Table 6: Historical mean In Vitro Irritation Score of the Negative Control (NaCl 0.9 %)from February 2015 until May 2018

	IVIS Negative Control - NaCl 0.9 %
Mean Value (MV)	1.09
Standard Deviation (SD)	0.76
MV- 2xSD	-0.44
MV+2xSD	2.62
Number of Replicates providing Historical Mean: 36

Table 7: Historical Data on Opacity and Permeability of the Negative Control (NaCl 0.9 %) from August 2017 until May 2018

Replicates Providing Historical Mean	Cornae No.	Opacity	Permeability	IVIS
		Change of Opacity Value	OD490 Value
1	1	0.234	0.008	1.49
	2	1.738	0.008
	3	0.800	0.098
2	1	0.978	0.019	2.52
	2	3.920	0.022
	3	1.617	0.028
3	1	-0.149	0.009	-0.50
	2	-0.415	0.015
	3	-1.427	0.009
4	1	0.776	0.008	0.92
	2	0.808	0.022
	3	0.418	0.020
5	1	0.035	0.010	0.35
	2	0.036	0.013
	3	0.466	0.012
6	1	1.030	0.017	0.79
	2	0.190	0.008
	3	0.640	0.009
7	1	0.714	0.024	0.74
	2	0.373	0.015
	3	0.371	0.012
8	1	1.034	0.013	0.94
	2	0.596	0.01
	3	0.623	0.015
9	1	0.789	0.006	0.79
	2	0.789	0.005
	3	0.543	0.005
Mean Value (MV)		0.649	0.016	0.893
Standard Deviation (SD)		0.894	0.017	0.813
MV- 2xSD		-1.140	-0.019	-0.732
MV+2xSD		2.438	0.051	2.519

 

EXPERIMENT 1 invalid

Table 1: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	2.92	3.03	0.12
2		2.61	2.99	0.38
3		2.61	2.73	0.11
MV		2.71	2.92	0.20
4	Positive Control	3.30	221.22	217.91	217.71
5		3.62	72.19	68.57	68.37
6		3.58	81.13	77.55	77.35
MV		3.50	124.85	121.35	121.14
7	Test Item	2.96	293.82	290.86	290.66
8		2.13	246.80	244.67	244.47
9		2.27	296.22	293.94	293.74
MV		2.45	278.95	276.49	276.29

MV = mean value

Table 2: Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.015
2		0.018
3		0.026
MV		0.020
4	Positive Control	2.625	2.605
5		3.210	3.190
6		2.945	2.925
MV		2.927	2.907
7	Test Item	1.385	1.365
8		1.061	1.041
9		1.040	1.020
MV		1.162	1.142
MV = mean value

Table 3: In vitro irritation score

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative Control	0.12	0.015
2		0.38	0.018
3		0.11	0.026
MV		0.20	0.020	0.50
4	Positive Control	217.71	2.605
5		68.37	3.190
6		77.35	2.925
MV		121.14	2.907	164.75
7	Test Item	290.66	1.365
8		244.47	1.041
9		293.74	1.020
MV		276.29	1.142	293.43
MV = mean value

Table 4: Historical mean in vitro irritation score of the positive control from February 2015 until January 2018

	IVIS Positive Control - Imidazole 20 %
Mean Value (MV)	122.57
Standard Deviation (SD)	17.33
MV- 2xSD	87.91
MV+2xSD	157.23
Number of Replicates providing Historical Mean: 33

Positive controls are updated after every single experiment or at least every 3 months.

Table 5: Historical data on opacity and permeability of the positive control (Imidazole 20 %) from August 2017 until January 2018

			Incubation: 240 min
	Number of Replicates	Cornea No.	Opacity		Permeability		IVIS
			Change of Opacity Value	Corrected Opacity Value	OD490 Value	Corrected OD490 Value
2017	1	4	122.785	121.861	0.662	0.624	133.420
		5	117.173	116.249	1.220	1.182
		6	102.655	101.731	2.260	2.222
	2	4	108.553	106.381	1.473	1.450	123.050
		5	79.491	77.319	2.465	2.442
		6	83.618	81.446	3.065	3.042
	3	4	55.644	56.308	2.200	2.189	92.540
		5	71.511	72.175	1.348	1.337
		6	65.148	65.812	2.040	2.029
	4	4	68.39	67.72	2.400	2.383	112.690
		5	70.88	70.21	2.810	2.793
		6	94.23	93.56	1.945	1.928
	5	4	70.53	70.35	1.296	1.284	102.440
		5	78.68	78.50	1.363	1.351
		6	91.69	91.51	1.840	1.828
2018	6	4	95.54	94.92	1.478	1.467	114.650
		5	83.58	82.96	1.500	1.489
		6	91.11	90.49	2.095	2.084
	Mean Value (MV)		86.178	85.528	1.859	1.840	113.132
	Standard Deviation (SD)		18.438	18.015	0.617	0.619	14.497
	MV- 2xSD		49.303	49.499	0.624	0.603	84.138
	MV+2xSD		123.053	121.558	3.094	3.078	142.126

Table 6: Historical mean in vitro irritation score of the negative control from February 2015 until January 2018

	IVIS Negative Control – NaCl 0.9 %
Mean Value (MV)	1.11
Standard Deviation (SD)	0.79
MV-2xSD	-0.47
MV+2xSD	2.70
Number of Replicates providing Historical Mean: 33

Negative controls are updated after every single experiment or at least every 3 months

Table 7: Historical data on opacity and permeability of the negative control (NaCl 0.9 %) from August 2017 until January 2018

			Incubation: 240 min
	Number of Replicates Providing Historical Mean	Cornae No.	Opacity	Permeability	IVIS
			Change of Opacity Value	OD490 Value
2017	1	1	0.234	0.008	1.49
		2	1.738	0.008
		3	0.800	0.098
	2	1	0.978	0.019	2.52
		2	3.920	0.022
		3	1.617	0.028
	3	1	-0.149	0.009	-0.50
		2	-0.415	0.015
		3	-1.427	0.009
	4	1	0.776	0.008	0.92
		2	0.808	0.022
		3	0.418	0.020
	5	1	0.035	0.010	0.35
		2	0.036	0.013
		3	0.466	0.012
2018	6	1	1.030	0.017	0.79
		2	0.190	0.008
		3	0.640	0.009
	Mean Value (MV)		0.650	0.019	0.928
	Standard Deviation (SD)		1.097	0.021	1.024
	MV- 2xSD		-1.543	-0.023	-1.120
	MV+2xSD		2.843	0.060	2.976

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The in vitro eye irritancy potential of Fatty acids, C9-C13-neo, barium salts cannot be assessed in the bovine corneal opacity and permeability assay due to residual test item on the cornea, confounding the transmission measurements. However, based on the qualitative visual appraisal of the cornea being markedly opaque, a conservative classification of Fatty acids, C9-C13-neo, barium salts being severely damaging to eye (H318) appears justified.
In conclusion, Fatty acids, C9-C13-neo, barium salts needs to be classified as serious eye damaging (EU CLP/ UN GHS Category 1).