Paraffin waxes (Fischer-Tropsch), isomerization

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-02-07 until 2022-07-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

26th June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

No. 1152/1010, 09 December 2010

Deviations:

no

Principles of method if other than guideline:

Commission Regulation (EU) No 2017/735 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 (Reach).
Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

GLP compliance:

yes (incl. QA statement)

Remarks:

date of GLP Certificate: 2019-10-23

Test material

Test material information

Constituent 1

Reference substance name:

Paraffin waxes (Fischer-Tropsch), isomerization

Cas Number:

2658498-20-3

Molecular formula:

C25H52 - C150H302

IUPAC Name:

Paraffin waxes (Fischer-Tropsch), isomerization

Details on test material:

- Purity: 100%
- Molecular weight: Not applicable (UVCB)
- Physical state / Appearance: Solid / white
- Expiry Date / Retest Date: Not indicated
- Storage Conditions: Room temperature
- Stability in Solvent: Not indicated

Specific details on test material used for the study:

Appearance: White, solid (waxy)
Storage Conditions: At room temperature

Test animals / tissue source

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany

Test system

Vehicle:

physiological saline

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The anterior compartment received the test item or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneas via open chamber method, respectively.

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneas per group (test item, negative controls, positive controls)

Details on study design:

Incubation Medium
The incubation medium consisted of MEM (Minimum Essential Medium), supplemented with 1.1 g sodium bicarbonate, 5 mL L-glutamine, 5 mL penicillin/streptomycin per 500 mL medium (final concentration of 100 units penicillin per mL medium, and 100 µg streptomycin per mL medium). Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS). MEM containing all supplements was called cMEM.
The OECD guideline 437 recommends the use of EMEM (Eagle’s minimum essential medium) which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.
Experimental Design and Study Conduct
Collectionof Bovine Eyes
Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Balanced Salt Solution)containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin)in the cooled slaughterhouse and during transportation on the same morning to the laboratoryusing a Styrofoam box. The corneas were isolated on the same day after delivery of the eyes.
PreparationofCorneas
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.The corneas were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.At the end of the incubation period, the medium was changed before basal opacity measurement (t0).
Only corneas with a value of the basal opacity < 7 were used. Sets of three corneas were used for treatment with the test item and for the negative and positive controls, respectively.

Exposure of the Corneas to the Test Groups
The corneas were distributed as follows:

The corneas were distributed as follows:

Groups Number of Corneas
1 Negative Control I 3
2 Negative Control II 3
3 Positive Control I 3
4 Positive Control II 3
5 Test Item 3

The anterior compartment received the test item or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneas via open chamber method, respectively. The corneas were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation period is 240 minutes.
After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Since the test item proved difficult to remove by the rinsing method, the front cover of the holder was opened, and the cornea was carefully washed using a gentle stream of incubation medium. Once the medium was free of the test item the corneas were given a final rinse with cMEM without phenol red. Fresh cMEM was added into both compartments and opacity was measured (t240).
Optical evaluation of the test item treated corneas with the unaided eye revealed no visible damage.

The opacity measurement is described in chapter 3.6. In the second step of the assay, permeability of the cornea was determined. The permeability measurement is described in chapter 3.7.
Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneas and displays a numerical opacity value. This value was recorded in a table. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
Permeability Determination
Following to the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
Data Recording
The data generated were recorded in the raw data file. The results are presented in tabular form, including experimental groups with the test item as well as negative and positive controls.
Data Evaluation
Opacity
The change of the opacity value of each treated cornea or of the positive and negative control corneas is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneas is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
Permeability
The corrected OD490 value of each cornea treated with positive control or test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.
In vitro Irritancy Score (IVIS) Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the corrected IVIS of the positive control and the test item:
IVIScorr = (opacity value – mean of opacity of negative control) + 15 x (permeability value – mean permeability of the negative control)
The mean IVIS value of each treated group was calculated from the individual IVIS values.
Depending on the IVIS score obtained, the test item is classified into the following Category according to OECD guideline 437:

IVIS GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made1
> 55 Category 1

The test method according to the OECD Guideline 437 does not allow for the evaluation of eye irritation (UN GHS Category 2). Further testing with other test methods will be required because the BCOP test shows a high rate (69%) of false positive results (“No UN GHS Category” predicted as “No prediction can be made”).

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Main experiment 1

Value:

3.25

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Main Experiment 2

Value:

0.66

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no category

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Main experiment 3

Value:

0.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no category

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
Main Experiment 1:
- Acceptance criteria met for negative control 1: fulfilled, no category (IVIS 1.51)
- Acceptance criteria met for negative control 2: fulfilled, no category (IVIS 1.45)
- Acceptance criteria met for positive control 1: fulfilled, category 1 (IVIS 99.79)
- Acceptance criteria met for positive control 2: fulfilled, category 1 (IVIS 105.26)

Main Experiment 2:
- Acceptance criteria met for negative control 1: fulfilled, no category (IVIS 1.28)
- Acceptance criteria met for negative control 2: fulfilled, no category (IVIS 1.32)
- Acceptance criteria met for positive control 1: fulfilled, category 1 (IVIS 88.36)
- Acceptance criteria met for positive control 2: fulfilled, category 1 (IVIS 93.22)

Main Experiment 3:
- Acceptance criteria met for negative control 1: fulfilled, no category (IVIS 0.87)
- Acceptance criteria met for negative control 2: fulfilled, no category (IVIS 1.88)
- Acceptance criteria met for positive control 1: fulfilled, category 1 (IVIS 91.10)
- Acceptance criteria met for positive control 2: fulfilled, category 1 (IVIS 106.55)

Any other information on results incl. tables

Results after 240 Minutes Treatment Time – first experiment

Test Group	Opacity value = Difference(t240-t0)of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Standard Deviation IVIS	Proposed Category
		Mean		Mean
Negative Control 1	0		0.076		1.14
Negative Control 1	0	0.33	0.093	0.079	1.40	1.51	0.44	No Category
Negative Control 1	1		0.067		2.01

 

Negative Control 2	0	0.085	1.28
Negative Control 2	1	0.075	2.13	1.45	0.60	No Category
Negative Control 2	0	0.064	0.96
Positive Control 1	80.67*	1.051*	96.44
Positive Control 1	89.67*	0.882*	102.90	99.79	3.24	Category 1
Positive Control 1	88.67*	0.758*	100.04
Positive Control 2	82.67*	1.071*	98.74
Positive Control 2	87.67*	1.355*	108.00	105.26	5.68	Category 1
Positive Control 2	86.67*	1.492*	109.05
Test Item	1.67*	0.013*	1.87
Test Item	2.67*	0.005*	2.75	3.25	1.69	No predictioncan be made
Test Item	4.67*	0.031*	5.14

^{*corrected values}

^{Negative Control 1: Saline}

^{Negative Control 2: deionised water}

^{Positive Control 1:10 % Benzalkonium chloride}

^{Positive Control 2: 20 % Imidazole}

 

Results after 240 Minutes Treatment Time – second experiment

Test Group	Opacity value = Difference(t240-t0)of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Standard Deviation IVIS	Proposed Category
		Mean		Mean
Negative Control 1	0		0.075		1.13
Negative Control 1	0	0.33	0.059	0.063	0.89	1.28	0.50	No Category
Negative Control 1	1		0.056		1.84

 

Negative Control 2	1	0.072	2.08
Negative Control 2	0	0.065	0.98	1.32	0.66	No Category
Negative Control 2	0	0.060	0.90
Positive Control 1	79.67*	0.451*	86.43
Positive Control 1	78.67*	0.417*	84.92	88.36	4.72	Category 1
Positive Control 1	88.67*	0.339*	93.75
Positive Control 2	80.67*	0.819*	92.95			Category 1
Positive Control 2	83.67*	0.842*	96.29	93.22	2.94
Positive Control 2	79.67*	0.718*	90.43
Test Item	0.00**	0.013*	0.19			No Category
Test Item	0.67*	0.023*	1.01	0.66	0.42
Test Item	0.67*	0.008*	0.78

^{*corrected values}

^{** Value was set to zero since the calculated value was negative}

^{Negative Control 1: Saline}

^{Negative Control 2: deionised water}

^{Positive Control 1:10 % Benzalkonium chloride}

^{Positive Control 2: 20 % Imidazole}

 

Results after 240 Minutes Treatment Time – third experiment

Test Group	Opacity value = Difference(t240-t0)of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Standard Deviation IVIS	Proposed Category
		Mean		Mean
Negative Control 1	0		0.059		0.89
Negative Control 1	0	0.00	0.057	0.058	0.86	0.87	0.02	No Category
Negative Control 1	0		0.057		0.86

 

Negative Control 2	1	0.083	2.25
Negative Control 2	1	0.073	2.10	1.88	0.51	No Category
Negative Control 2	0	0.087	1.31
Positive Control 1	71.00*	0.826*	83.40
Positive Control 1	83.00*	0.743*	94.15	91.10	6.72	Category 1
Positive Control 1	81.00*	0.984*	95.77
Positive Control 2	84.00*	1.227*	102.41
Positive Control 2	88.00*	1.409*	109.14	106.55	3.62	Category 1
Positive Control 2	91.00*	1.139*	108.09
Test Item	0.00*	0.024*	0.37
Test Item	0.00*	0.010*	0.16	0.30	0.12	No Category
Test Item	0.00*	0.024*	0.37

^{*corrected values}

^{Negative Control 1: Saline}

^{Negative Control 2: deionised water}

^{Positive Control 1:10 % Benzalkonium chloride}

^{Positive Control 2: 20 % Imidazole}

Applicant's summary and conclusion

Interpretation of results:

other: no categorisation

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, 'Paraffin waxes (Fischer-Tropsch), isomerization' is not categorized (EU CLP/GHS No Category).

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' by means of the BCOP assay using fresh bovine corneas.

The test was performed threefold since in the first run the IVIS was a borderline result (IVIS of the single corneas were 1.87, 2.75, 5.14, mean IVIS: 3.25). According to OECD 437 a second experiment was performed. Since the second experiment showed discordant results to the first one a confirmatory third experiment was performed.

After a first opacity measurement of the fresh bovine corneas (t0), the sample of the test item 20% (w/v) in saline (0.9% (w/v) NaCl in deionised water) as an inhomogeneous suspension as well as the positive and the negative controls were each applied to different corneas fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t240).

After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative controls (saline and deion. water), neither an increase of opacity nor permeability of the corneas could be observed.

The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas corresponding to a classification as serious eye damage (EU CLP/GHS Category 1) as well as the positive control 20 % (w/v) imidazole in saline.

Relative to the negative control, the test item 'Paraffin waxes (Fischer-Tropsch), isomerization' did not cause a relevant increase of the corneal opacity or permeability in the second and third experiment. The calculated mean in vitro irritancy score was 0.66 (second experiment) and 0.30 (third experiment).

According to OECD 437 the test item 'Paraffin waxes (Fischer-Tropsch), isomerization' is not categorized (EU CLP/GHS No Category).