Alkali metal tetrakis (perfluoro-alcoholato)metallate

Endpoint summary

Description of key information

The substance was tested in two in vitro studies for skin irritation/corrosion. Whereas a study according to OECD Guideline 431 and EU Method B.40-BIS (Determination of Skin Corrosion Potential of AM(pfa)4 in the Reconstructed Human Epidermis (RHE) Test Method) showed that the substance is non-corrosive a further study according to OECD Guideline 439 and EU-Method B.46 (Determination of Skin Irritation Potential of AM(pfa)4 in the Reconstructed human Epidermis (RhE) Test Method) showed that the substance is considered at least as irritant to skin. Thus, AM(pfa)4 is considered as an irritant to skin, category 2, according to CLP (Regulation EC No. 1272/2008).

Furthermore, the substance was tested in two in vitro studies for eye irritation/damage. Whereas a study according to OECD Guideline 437 and EU Method B.47 (Bovine Corneal Opacity and Permeability (BCOP) Test Method) showed that the substance is non-damaging to eyes a further study according to OECD Guideline 492 (Determination of Eye Hazard Potential of AM(pfa)4 using the EpiOcular™ Reconstructed human Cornea-like Epithelium (RhCE) test method) showed that the substance is considered at least as irritant to eyes. Thus, AM(pfa)4 is considered as an irritant to eye, category 2, according to CLP (Regulation EC No. 1272/2008).

Information on respiratory irritation is not available.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

18.6.2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EpiDerm™ tissues derived from MatTek Corporation, Lot# 30863, keratinocyte strain 00267

Justification for test system used:

The skin corrosion test refers to the production of irreversible tissue damage following the application of a test material on a reconstructed human skin model. It allows the identification of corrosive chemical substances and mixtures.
The test item is applied topically to a three-dimensional human skin model, comprising of non-transformed, human-derived epidermal keratinocytes, which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo.
The principle of the human skin model assay is based on the hypothesis that corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion and are cytotoxic to the underlying cell layers.
Corrosive chemicals are identified by their ability to decrease cell viability. The viability is measured by enzymatic conversion of the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) into a blue formazan salt, that is quantitatively measured after extraction from tissues.

Vehicle:

water

Details on test system:

The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.
EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava (Designation of the kit: EPI-200-SCT, Batch: 30863)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

in the 3 minute exposure experiment two tissues were tested with 25.9 mg and 25.2 mg test substance; for the 1 hour exposure experiment also two tissues were exposed to 26.2 mg and 26.3 mg test substance, respectively.

Duration of treatment / exposure:

3 minutes and 1 hour exposure

Duration of post-treatment incubation (if applicable):

After the respective incubation time (“3 minutes” and “1 hour”) at 37 ± 1 °C and 5.0 ±1% CO2, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with DPBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. After transfer of all inserts, they were immediately moved to the wells containing MTT medium, blotting the bottom with cellulose tissue again before setting the insert into the MTT well. The tissues were incubated with MTT solution for 3 hours at 37 ±1 °C and 5.0 ±1% CO2.
After this time, the MTT solution was aspirated and replaced by DPBS. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipetted, taking care to reach the upper rim of the insert. The plate was then shaken for 2 hours at room temperature.

Number of replicates:

Two tissues per exposure time were tested

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item, 3 minutes incubation

Value:

109

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control, 3 minutes incubation

Value:

20.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item, 1 hour incubation

Value:

79.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive control, 1 hour incubation

Value:

4.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Validity
The criterion for optical density of the negative control (≥ 0.8 and ≤ 2.8) was fulfilled: optical density was 1.6 (3 minutes) resp. 1.7 (1 hour).
The positive control showed clear corrosive effects. The criterion for the viability of the 1 hour experiment, expressed as % of the negative control (< 15%), was fulfilled, too. The mean value of relative tissue viability was 4.9%.
The values for negative control and for positive control were within the range of historical data of the test facility.
Therefore, the experiment is considered valid.

Measured Values

As blank, the optical density of isopropanol was measured in 12 wells of the 96-well-plate. The measured values and their mean are given as follows:

Absorbance: 0.035, 0.036, 0.035, 0.038, 0.035, 0.035, 0.035, 0.035, 0.035, 0.034, 0.034, 0.035, Mean of 12 values: 0.035

 

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values (OD 570 nm)

Incubation	Negative Control		Test Item		Positive Control
	Tissue 1	Tissue 2	Tissue 1	Tissue 2	Tissue 1	Tissue 2
3 min	1.575	1.686	1.753	1.798	0.354	0.376
	1.574	1.689	1.736	1.793	0.353	0.377
	1.567	1.668	1.747	1.794	0.351	0.379
1 h	1.760	1.623	1.397	1.302	0.118	0.114
	1.748	1.619	1.385	1.299	0.117	0.115
	1.750	1.616	1.381	1.294	0.118	0.114

 

From the measured absorbances, the mean absorbance of isopropanol was subtracted. The corrected mean and relative standard deviation (RSD) of the two tissues were also calculated.

Mean Absorbance Values of the 3 Minutes Experiment

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.537	1.710	0.318
Mean – blank (tissue 2)	1.646	1.760	0.342
Mean of the two tissues	1.591	1.735	0.330
RSD	4.8%	2.0%	5.3%

 

Mean Absorbance Values of the 1 h Experiment

Designation	Negative Control	Test Item	Positive Control
Mean – blank (tissue 1)	1.718	1.353	0.083
Mean – blank (tissue 2)	1.584	1.263	0.079
Mean of the two tissues	1.651	1.308	0.081
RSD	5.7%	4.8%	2.9%

 

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of mean tissue viability were calculated in comparison to the mean of the negative controls:

% Tissue Viability

Test Item	Positive Control	Incubation
109.0%	20.7%	3 min
79.2%	4.9%	1 h

 

Corrosivity of the Test Item

The mean value of relative tissue viability of the test item was increased to 109.0% after 3 minutes treatment. This value is above the threshold for corrosivity (50%). After 1 hour treatment, the mean value of relative tissue viability of the test item was reduced to 79.2%, lying above the threshold for corrosivity (15%). Therefore, the test item is considered as non-corrosive to skin in this study.

Interpretation of results:

other: substance is non-corrosive to skin

Conclusions:

The test item AM(pfa)4 is considered non-corrosive to skin.

Executive summary:

One valid experiment was performed.

Two tissues of the human skin model EpiDerm™ were treated with the test item for 3 minutes and 1 hour, respectively. The test item was applied to each tissue and spread to match the tissue size.

Demineralised water was used as negative control and 8 M KOH was used as positive control.

After treatment, the respective substance was rinsed from the tissues. Then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to a blue formazan. Formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for both treatment intervals; thus, showing the quality of the tissues. The OD was 1.6 (3 minutes experiment) and 1.7 (1 hour experiment).

The positive control showed clear corrosive effects for both treatment intervals. The mean relative tissue viability value was reduced to 4.9% for the 1 hour treatment.

After 3 minutes treatment with the test item, the mean value of relative tissue viability was increased to 109.0 %. This value is above the threshold for corrosion potential (50%). After 1 hour treatment, the mean value of relative tissue viability was reduced to 79.2%. This value too is above the threshold for corrosion potential (15%).

 

Thus, under the conditions of this study, the test item AM(pfa)4 is considered non-corrosive to skin in the Reconstructed Human Epidermis (RHE) Test method.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2019 - Jan. 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no.: 931-EMP-19004

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava. Designation of the kit: EPI-200-SIT

Details on test system:

The test system is a commercially available EpiDerm™-Kit, procured by MatTek. The EpiDerm™ tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDerm™ tissues are cultured on specially pre-pared cell culture inserts. Designation of the kit: EPI-200-SIT, Batch no.: 30842

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25.2 mg were applied for each replicate

Duration of treatment / exposure:

All working steps were performed under sterile conditions. For each treatment group (negative control, test item and positive control) a 6-well-plate was prepared with 0.9 mL assay medium in 3 of the 6 wells (upper row). The tissues were inspected for viability. Then, the tissues were transferred into the wells, which contain medium by using sterile forceps and placed into the incubator at 37 ±1 °C and 5 ±1% CO2 and ≥ 95% relative humidity for 1 hour. After 1 hour pre-incubation, the other 3 wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 ±1 °C and 5.0 ±1% CO2 and ≥ 95% relative humidity for 19 hours and 55 minutes.

Duration of post-treatment incubation (if applicable):

One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate was used as positive control; each tissue was treated with 30 µL 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue surface. One plate was used for treatment with the test item: The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size. Three replicates of 25.2 mg test item each were assessed. Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ±1 °C and 5.0 ±1% CO2 and ≥ 95% relative humidity. 1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals. After rinsing thoroughly with DPBS, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The surface of the inserts was then carefully dried with a sterile cotton tipped swab. Then, the tissues were set in the incubator for 23 hours at 37 ±1 °C and 5.0 ±1% CO2 and ≥ 95% relative humidity.

Number of replicates:

3 test item replicates were tested in duplicates, besides negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 1

Value:

5.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 2

Value:

5.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 3

Value:

6.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (tissues 1 - 3)

Value:

5.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

As blank, the optical density of isopropanol was measured in 8 wells of the 96-well-plate. The measured values and their mean are given in the following table:

Absorbance values blank isopropanol (OD 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.037	0.036	0.036	0.035	0.035	0.034	0.035	0.035	0.035

 

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values negative control, test item and positive control (OD 570 nm)

Designation	Measurement	Negative Control	AM(pfa)4	Positive Control
Tissue 1	1	1.251	0.101	0.078
	2	1.273	0.100	0.079
Tissue 2	1	1.257	0.096	0.074
	2	1.258	0.100	0.075
Tissue 3	1	1.213	0.115	0.073
	2	1.238	0.117	0.074

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table 9.1-a. The mean of the three tissues was also calculated.

Mean Absorbance Values

Designation	Negative Control	AM(pfa)4	Positive Control
Mean – blank (tissue 1)	1.227	0.066	0.044
Mean – blank (tissue 2)	1.223	0.063	0.040
Mean – blank (tissue 3)	1.191	0.081	0.039
Mean of the three tissues	1.214	0.070	0.041

 

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control:

% Tissue Viability

Designation	AM(pfa)4	Positive Control
% Tissue viability (tissue 1)	5.4%	3.6%
% Tissue viability (tissue 2)	5.2%	3.3%
% Tissue viability (tissue 3)	6.7%	3.2%
% Tissue viability (mean)	5.8%	3.4%
± SD of mean tissue viability (%)	0.8%	0.2%

 

Assessment and Validity

Skin Irritation Potential of the Test Item: The mean value of relative tissue viability of the test item was reduced to 5.8% after the treatment with the test item. This value is below the threshold for skin irritation (50%). Therefore, the test item is considered at least as irritant to skin.

Validity and Acceptability

Validity criteria and results are stated in the following table:

Validity

Criterion	Demanded	Found
OD of negative control	≥ 0.8 and ≤ 2.8	1.2
% tissue viability of positive control SDS	≤ 20% of negative control	3.4%
SD of mean viability of the tissue replicates (%)	≤ 18%	1.6% (negative control) 0.2% (positive control) 0.8% (test item)

 

All validity criteria were met. Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Interpretation of results:

study cannot be used for classification

Remarks:

at least as irritant to skin

Conclusions:

The test item AM(pfa)4 is considered at least as irritant to skin. After the treatment, the mean value of relative tissue viability was reduced to 5.8%. This value is below the threshold for skin irritation (50%).

Executive summary:

In this study two experiments were performed. The first experiment (Exp.1) was not valid, because the criterion for the negative control was not fulfilled, the mean optical density was 0.6, but required is ≥ 0.8 and ≤ 2.8. This experiment is not reported, but the raw data were kept in the test facility in the GLP-archive. The second experiment (Exp. 1b) was valid and the results are reported here.

Three tissues of the human skin model EpiDerm™ were treated with the test item for 60 minutes. The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm²; as indicated by the supplier). DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, the OD was 1.2. The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 3.4% (required: ≤ 20%). The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%).

After the treatment with the test item, the mean value of relative tissue viability was reduced to 5.8%. This value is below the threshold for skin irritation potential (50%). Test items that induce values below the threshold of 50% are considered at least irritant to skin.

The OECD guideline 439 addresses the human health endpoint skin irritation. Therefore, further testing on skin corrosion potential with another suitable in vitro study (e.g. OECD 431) may be required.

Under the conditions of the test, the test item AM(pfa)4 is considered at least as irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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)

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.

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.

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

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

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

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.

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019 - 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18.6.2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch no.: 931-EMP-19004

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Commercially available EpiOcular™ kit: The EpiOcular™ tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular™ tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm².
EpiOcular™ tissues as used were procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia (Designation of the kit: OCL-212-EIT, delivery on 10. Dec. 2019, Batch no.: 30638).

Vehicle:

unchanged (no vehicle)

Controls:

other: negative control: sterile demineralised water; positive control: methyl acetate (CAS No. 79-20-9)

Amount / concentration applied:

50.4 and 50.7 mg (two replicates)

Duration of post- treatment incubation (in vitro):

6 hours incubation

Number of animals or in vitro replicates:

2 replicates were measured

Details on study design:

CHEMICALS / MEDIA USED
MTT solution: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT), which can be re-duced to a blue formazan, prepared by LAUS GmbH. A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of 2 mL at – 20 ±5 °C. 2 mL of the stock solution were thawed and diluted with 8 mL of assay medium (resulting in 1 mg/mL). This MTT-solution with the concentration of 1 mg/mL was used in the test. For the pre-test (testing the ability of direct MTT reduction), the stock solution was thawed and diluted with serum-free MEM directly before use. For the main test, the stock solution was thawed and diluted with assay medium directly before use.
DPBS-Buffer: “Dulbecco`s Phosphate Buffered Saline” (DPBS) was used for the rinsing of the tissues. A subset was procured from MatTek In Vitro Life Science Laboratories (batch no.: 112719ISE: containing 0.2 g KCl, 0.2 g KH2PO4, 8.0 g NaCl, 2.16 g Na2HPO4 * 7H2O, ad to 1 L H2O); the other subset was prepared by LAUS GmbH (batch no.: T20190910, containing 0.2 g KCl, 0.2 g KH2PO4, 8.0 g NaCl, 1.44 g Na2HPO4 * 2H2O, ad to 1 L H2O). The buffer which was procured from MatTek Corporation was used for rinsing the test item from the tissues. The buffer which was prepared by LAUS GmbH was only used for preparing the MTT concentrate.
MEM with Phenol Red for Pre-Test: Serum-free MEM (Minimum Essential Medium), procured from Life Technologies GmbH, batch no.: 2078370.
Assay Medium for Main Test: Serum-free DMEM (Dulbecco’s Modified Eagle’s Medium), procured from MatTek, batch no.: 120919ISA.
Isopropanol: CH3-CH(OH)-CH3, p.A., 99.9 %, batch no.: 457264070, used as extracting solvent for formazan.
Test Vessels: All vessels used are made of sterilized plastic. The following vessels were used: 96-well-plates, 24-well plates, 12-well plates, and 6-well plates.
Demonstration of Proficiency
The validity of the EpiOcularTM test at LAUS GmbH was demonstrated in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcularTM test was demonstrated.
PERFORMANCE OF THE STUDY
Pre-Tests
Assessment of Direct Reduction of MTT by the Test Item: The test item was tested for the ability of direct MTT reduction. To test for this ability, 51.6 mg of the solid test item were added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ±1 °C, 5.0 ±1 % CO2 and ≥ 95% relative humidity for 3 hours. 1 mL of MTT solution plus 50 µL of H2O demin. was used as negative control. The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place and no data correction was necessary.
Assessment of Coloured or Staining Test Items: 50.7 mg of the test item were added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 2 hours at room temperature. Then, two 200 µL aliquots of the resulting solution and two 200 µL aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm. After subtraction of the mean OD for isopropanol, the mean OD of the test item solution was 0.0035 (≤ 0.08). Therefore, the main test was performed without colourant controls.
Main Test
Preparations: On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use. The assay medium was warmed in the water bath to 37 ±1 °C.
6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ±1 °C, 5 ±1 % CO2 and ≥ 95% relative humidity for 1 hour. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ±1 °C, 5 ±1 % CO2 and ≥ 95% relative humidity for 16 hours and 27 minutes.
Exposure and Post-Treatment: After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and the tissues were incubated at 37 ±1 °C, 5 ±1 % CO2 and ≥ 95% relative humidity for 30 minutes. After that, 50 µL of the controls and a defined amount of the test item (50.4 mg in replicate 1 and 50.7 mg in replicate 2) were applied in duplicate in one-minute-intervals.
At the beginning of each experiment (application of negative controls), a stopwatch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ±1 °C, 5 ±1 % CO2 and ≥ 95% relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature. After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ±1 °C, 5 ±1 % CO2 and ≥ 95% relative humidity. After the post-treatment incubation, the MTT assay was performed.
MTT Assay and Extraction: A 24-well-plate was prepared with 300 µL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solution. The plate was incubated for 195 minutes at 37 ±1 °C, 5 ±1 % CO2 and ≥ 95% relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.
Measurement: The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.
EVALUATION
The values of the 96-plate-reader were transferred into a validated spreadsheet (Microsoft Excel®).
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.
Calculations
- Calculation of mean OD of the blank isopropanol (ODBlk)
- Subtraction of mean ODBlk of each value of the same experiment (corrected values)
- Calculation of mean OD of the two replicates for each tissue
- Calculation of mean OD of the two relating tissues for controls and test item
Note: Corrected OD value of negative control corresponds to 100 % viability. To calculate the relative tissue viability, the following equation was used: % Viability =100 * OD(corrected of test item or positive control) / OD(corrected of mean negative control)

Irritation parameter:

other: absorbance value (corrected)

Run / experiment:

replicate 1

Value:

0.021

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: absorbance value (corrected)

Run / experiment:

replicate 2

Value:

0.018

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Viability [%]

Run / experiment:

replicate 1

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Viability [%]

Run / experiment:

replicate 2

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All validity criteria were met as follows:
- Mean OD of negative control: > 0.8 and < 2.8; found: 1.9
- % mean relative viability of positive control: < 50% of negative control; found: 30.7%
- Variation within replicates: < 20%: 0.8% (negative control), 2.7% (positive control), 0.2% (test item)
Therefore, the experiment is considered valid.

FINDINGS AND RESULTS

Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.031	0.032	0.032	0.033	0.032	0.033	0.032	0.033	0.032

 

The absorbance values of negative control, test item and positive control are given in the following table:

Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	AM(pfa)4
Tissue 1	1	1.905	0.564	0.054
	2	1.908	0.597	0.052
Tissue 2	1	1.882	0.616	0.050
	2	1.900	0.644	0.050

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table above (= corrected values).

Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	AM(pfa)4
Mean – blank (Tissue 1)	1.875	0.549	0.021
Mean – blank (Tissue 2)	1.859	0.598	0.018

 

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of tissue viabil-ity were calculated in comparison to the negative control:

% Viability Positive Control and Test Item

Designation	Positive Control	AM(pfa)4
% Viability (Tissue 1)	29.4%	1.1%
% Viability (Tissue 2)	32.0%	1.0%
% Viability Mean	30.7%	1.0%

 

Assessment

Eye hazard potential is assessed using the criteria as given in the test guideline:

Viability > 60 % is considered “non eye irritant” (no category according to GHS)

Viability ≤ 60 % is considered “at least eye irritant” (no prediction can be made for category 1 or 2)

Interpretation of results:

other: category 1 or 2, based on GHS criteria

Conclusions:

Under the conditions of the test, AM(pfa)4 is considered either eye irritant or inducing serious eye damage in the EpiOcular™ Eye Irritation Test.

Executive summary:

The test item AM(pfa)4 was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours. The solid test item was applied to two tissue replicates.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control and methyl acetate was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and < 2.8, OD was 1.9. The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 30.7% (< 50%). The variation within tissue replicates of the controls and the test item was acceptable (< 20%). Therefore, the result of the test is considered valid.

After treatment with the test item, the mean value of relative tissue viability was 1.0%.

This value is below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage. According to the OECD Guideline 492, the EpiOcular™ Eye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required.

Under the conditions of the test, AM(pfa)4 is considered either eye irritant or inducing serious eye damage in the EpiOcular™ Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was tested in two in vitro studies for skin irritation/corrosion. Based on the outcome of these studies, AM(pfa)4 is considered as an irritant to skin, category 2, according to CLP (Regulation EC No. 1272/2008). As conclusion from two in vitro studies for effects to eyes, it was found that te substance is not considered damaging to eyes, but irritant to eyes. Thus, the substance AM(pfa)4 is considered as an irritant to eye, category 2, according to CLP (Regulation EC No. 1272/2008).