Alkali metal tetrakis (perfluoro-alcoholato)metallate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

9.10.2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

Test material was sublimed for purification.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The test method uses isolated corneas from the eyes of freshly slaughtered cattle.

Test system

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL test item suspension (tested as a suspension with a concentration of 20% in Hank’s Balanced Salt Solution (HBSS); 10-fold concentrated, which was previously diluted in demin. water (1:10)

Duration of treatment / exposure:

Exposure time of corneas was 4 hours.

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

Incubation for 90 minutes at 32 ±1 °C in a horizontal position

Number of animals or in vitro replicates:

Three replicates per treatment group (negative control, test item and positive control)

Details on study design:

This in vitro study was performed to assess the corneal damage potential of AM(pfa)4 by quantitative measurements of changes in opacity and permeability in a bovine cornea. The BCOP test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. Both measurements are used to calculate an “In Vitro Irritancy Score (IVIS)”, which is used to classify the test item in the UN Globally Harmonised System (GHS).
The BCOP test method uses isolated corneas from the eyes of freshly slaughtered cattle. The test item is applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. Corneal opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative controls (HBSS)

Value:

-0.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item (AM(pfa)4)

Value:

24.94

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control (20% imidazole solution)

Value:

105.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Opacity and Permeability Values

The illuminance (unit: LUX) values which were measured before and after exposure are given in the following table:

Illuminance Values

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Measured values [I] before exposure	1025	1006	1009	1028	1014	1015	1004	1007	999
Measured values [I] after exposure	1024	1010	1023	587	690	669	349	337	346

Rep. = Replicate

 

The values in the following tables present the calculated opacity values, according to evaluation scheme:

Opacity Values Negative Control

Parameter	Negative Control
	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	3.10	3.90	3.77
Opacity after exposure	3.14	3.73	3.18
Opacity Difference	0.04	-0.17	-0.59
Mean Opacity Difference	-0.24

Rep. = Replicate

 

Opacity Values Test Item and Positive Control

Parameter	Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
Opacity before exposure	2.98	3.56	3.52	3.99	3.86	4.21
Opacity after exposure	34.83	23.74	25.72	85.46	89.90	86.54
Opacity Difference	31.85	20.18	22.21	81.47	86.04	82.33
Opacity Difference corrected	32.09	20.42	22.45	81.71	86.28	82.57
Mean Opacity Difference corrected	24.99			83.52

Rep. = Replicate

For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:

Optical density at 492 nm of Blank

Parameter	cMEM without phenol red
1. Measurement	0.038
2. Measurement	0.037
3. Measurement	0.034
Mean	0.036

 

Optical density at 492 nm of Negative Control, Test Item and Positive Control

Parameter	Negative Control			Test Item			Positive Control
	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.	1. Rep.	2. Rep.	3. Rep.
1.Mesurement	0.043	0.041	0.045	0.040	0.042	0.037	1.485	1.040	1.967
2.Measurement	0.043	0.042	0.044	0.041	0.041	0.038	1.474	1.033	1.930
3.Measurement	0.043	0.043	0.044	0.040	0.042	0.038	1.471	1.027	1.919
1.Measurement – blank	0.0067	0.0047	0.0087	0.0037	0.0057	0.0007	1.4487	1.0037	1.9307
2.Measurement – blank	0.0067	0.0057	0.0077	0.0047	0.0047	0.0017	1.4377	0.9967	1.8937
3.Measurement – blank	0.0067	0.0067	0.0077	0.0037	0.0057	0.0017	1.4347	0.9907	1.8827
Mean of each replicate	0.0067	0.0057	0.0080	0.0040	0.0053	0.0013	1.4403	0.9970	1.9023
Mean of the 3 replicates	0.0068			--			--
Corrected	--	--	--	-0.0028	-0.0014	-0.0054	1.4336	0.9902	1.8956
Corrected mean of the 3 replicates	--			-0.0032			1.4398

Rep. = Replicate

IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Test Group	IVIS	Mean IVIS	Relative Standard Deviation IVIS
Negative Control HBSS	0.14	-0.14	223.23%
	-0.09
	-0.47
Test Item LiAl(pftb)4	32.05	24.94	25.00%
	20.40
	22.37
Positive Control 20% imidazole solution	103.21	105.12	4.95%
	101.14
	111.01

Note: the high relative standard deviation of the IVIS of the negative control is due to mathematical reasons, as the respective means are very small.

Validity

According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.

The mean IVIS of the negative control has to show an IVIS ≤3.

The validity criteria and findings are given in the following table:

Validity

Parameter	Criterion	Found	Assessment
Mean IVIS of negative control HBSS	≤ 3	-0.14	ok
Mean IVIS of positive control 20% imidazole solution	75.90 – 144.68	105.12	ok

 

All relevant values for negative and positive control were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Assessment

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP test only. In this case no prediction can be made.

Classification Scheme

IVIS	UN GHS
≤ 3	No category
> 3 and ≤ 55	No prediction can be made
> 55	Eye damage Category I

In the negative control, no signs of eye irritation were observed.

The positive control induced serious eye damage, which would be classified as GHS category I.

The test item AM(pfa)4 showed effects on the cornea of the bovine eye. The calculated mean IVIS (In Vitro Irritancy Score) was 24.94. The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.

Applicant's summary and conclusion

Interpretation of results:

other: study shows that substance has effects to eyes but is not considered eye damaging.

Conclusions:

According to the outcome of this study the substance AM(pfa)4 is not considered as severe eye damaging, but showed effects on the cornea of the bovine eye.

Executive summary:

This in vitro study was performed to assess corneal damage potential of AM(pfa)4 by quantitative measurements of changes in opacity and permeability in a bovine cornea. As test system bovine corneas were used derived from slaughtered cattle that were between 12 and 60 months old. The test item AM(pfa)4 was diluted to a concentration of 20% in HBSS. The suspension was applied onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ±1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ±1 °C. After removal of the test item, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated mean IVIS (In Vitro Irritancy Score) was -0.14. 20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated mean IVIS was 105.12.

Under the conditions of this study, the test item AM(pfa)4 showed effects on the cornea of the bovine eye. The calculated mean IVIS was 24.94.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP study only. In this case no prediction can be made. However, the substance is not considered as severe eye damaging according to the results in this study.