Paraffin waxes (Fischer-Tropsch), isomerization

Endpoint summary

Description of key information

Skin irritation / corrosion

- reliable OECD 439 in vitro study on registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization', result: not irritating;

- reliable OECD 404 in vivo study on closely related substance GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0) covering the entire low molecular weight fraction of the registration substance up to C50, result: not irritating;

- reliable OECD 404 in vivo study on closely related substance GTL waxy raffinate (C15-50; CAS 848301-87-1, EC 482-130-1) covering the entire low molecular weight fraction of the registration substance up to C50, result: not irritating;

- supporting data on further closely related substances also confirm that the registration substance should not be irritating when applied to the skin of rabbits.

 

Eye irritation

- reliable OECD 437 ("BCOP") in vitro study on registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization', result: not irritating;

- reliable OECD 404 in vivo study on closely related substance GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0), covering the entire low molecular weight fraction of the registration substance up to C50, result: not irritating;

- reliable OECD 404 in vivo study on closely related substance GTL waxy raffinate (C15-50; CAS 848301-87-1, EC 482-130-1) covering the entire low molecular weight fraction of the registration substance up to C50, result: not irritating;

- supporting data on further closely related substances also confirm that the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' should not be irritating when applied to the eye of rabbits.

 

Respiratory irritation

Taking into account the very low vapour pressure of the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization', exposure via the inhalation route is unlikely and it is therefore considered justified to omit this endpoint information.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Remarks:

Skin Irritation Test

Adequacy of study:

key study

Study period:

2022-02-10 until 2022-03-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: • MatTek Corporation Protocol: In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT); Version 12/04/2020. • MatTek Corporation Protocol: EpiOcularTM Eye Irritation

Version / remarks:

Note:
The Pre-Test (Assessment of Colored or Staining Materials and Assessment of Direct Test Article Reduction by MTT) was performed according to the MatTek Protocol “EpiOcularTM Eye Irritation Test.
Reason:
The OECD TG 439 requires the evaluation of colored or staining materials spectrophotometrically but does not state the evaluation criteria. These criteria are missing in the MatTek protocol “In vitro EpiDermTM Skin Irritation Test”, too. But the identical scientific hypothesis is to clarify in the Eye Irritation Test and there the requirements are described.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UN GHS

Version / remarks:

published 2003, last (8th revision) 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Reconstructed Human Epidermis Test Methods (14 June 2021), and as described in detail in the Protocol for:
In vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) for use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200).

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

06 July 2012

Deviations:

no

GLP compliance:

yes

Remarks:

Date of GLP Certificate: 2019-10-23

Specific details on test material used for the study:

Appearance: White, solid (waxy)
Storage Conditions: At room temperature
Stability in Solvent: Not indicated

Test system:

human skin model

Source species:

other: Standard Assay Kit and MTT-100 kit were purchased from MatTek Corporation (82105 Bratislava, Slovakia, Lot No.: 36129).

Justification for test system used:

Dermal irritation is generally defined as "the production of reversible inflammatory changes in the skin". The potential for chemical induced skin irritation is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. It is usually determined in the in vivo Draize rabbit skin irritation test as described in OECD guideline 404. Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international prevalidation study performed by ECVAM, the in vitro skin irritation test using the human skin model EpiDerm™ and EpiSkin™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.

Vehicle:

unchanged (no vehicle)

Details on test system:

In vitro Skin Irritation Test (OECD 439)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):25 +/- 2 mg (39.7 mg/cm2 according to guideline)

NEGATIVE CONTROL
- Concentration (if solution): 30 µL DPBS

POSITIVE CONTROL
- Concentration (if solution): 5% SDS solution in deionised water (MatTek)

Duration of treatment / exposure:

60 minutes

Number of replicates:

Three tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item

Value:

100.08

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

according to UN GHS and EU CLP regulations

Main Experiment: Results after treatment with test item and controls

Treatment Group	Tissue No.	Well 1 OD 570 nm	Well 2 OD 570 nm	Well 3 OD 570 nm	Mean OD of 3 Wells	Mean OD of 3 Wells blank corrected	Mean OD of 3 tissues	Rel. Viability [%] Tissue 1, 2 + 3	Standard Deviation	Mean Rel. Viability [%]
Treatment Group	Tissue No.	0.037
Blank		1.580	0.037	0.037	0.037
Negative Control	1	1.625	1.537	1.561	1.559	1.522		97.658
	2	1.658	1.596	1.590	1.604	1.567	1.559	100.530	2.1	100.0
	3	0.125	1.606	1.607	1.624	1.587		101.813
Positive Control	1	0.100	0.110	0.109	0.115	0.078		5.004
	2	0.106	0.094	0.092	0.095	0.058	0.066	3.742	0.7	4.25
	3	1.575	0.097	0.094	0.099	0.062		3.997
Test Item	1	1.626	1.567	1.565	1.569	1.532		98.297
	2	1.607	1.609	1.611	1.615	1.579	1.560	101.272	1.6	100.08
	3				1.606	1.569		100.669

^{OD: optical density}

Interpretation of results:

other: EU-GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the registration substance is non-irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the skin irritation potential of the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' by means of the Human Skin Model Test.

The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic color was not intensive and the OD of the test item in deionised water or isopropanol at 570 nm after blank correction was < 0.08. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed.

Each three tissues of the human skin model EpiDerm^™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a decrease in the viability compared with the negative control to 4.25%, thus ensuring the validity of the test system.

The standard deviations between the three tissue percentage viability values of each group (test item, positive and negative controls) in the main test were below 18 (threshold according to OECD 439: ≤ 18), thus ensuring the validity of the study.

After treatment with the test item the mean viability value was 100.08% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November 2007 and 16 November 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: August 2007; Date of certificate: October 2007

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Limited, Bicester, Oxon, UK.
- Age at study initiation: twelve to twenty weeks old.
- Weight at study initiation: 2.0 to 3.5 kg.
- Housing:The animals were individually housed in suspended cages.
- Diet (e.g. ad libitum): Free access to mains drinking water and food (Certified Rabbit Diet) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Water (e.g. ad libitum):Free access to mains drinking water and food (Certified Rabbit Diet) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Acclimation period: Acclimatisation period of at least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): The rate of air exchange was at least fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES: From:0 To:3

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A quantity of 0.5 ml of the test material was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin.
- Concentration (if solution): No Data

VEHICLE
- Amount(s) applied (volume or weight with unit): Not applicable
- Concentration (if solution):Not applicable
- Lot/batch no. (if required): Not applicable
- Purity:Not applicable

Duration of treatment / exposure:

5 hours

Observation period:

Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored.

Number of animals:

Three rabbits

Details on study design:

TEST SITE
- Area of exposure: On the day before the test each of a group of three rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. On the day of the test a suitable test site was selected on the back of each rabbit.
- % coverage: 2.5 cm x 2.5 cm
- Type of wrap if used: The patch was secured in position with a strip of surgical adhesive tape.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): Five hours after application the corset and patches were removed from each animal and any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure:Five hours after application.


SCORING SYSTEM:
EVALUATION OF SKIN REACTIONS
Erythema and Eschar Formation Value
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema 4

Oedema Formation
Value
No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

An isolated incident of very slight erythema was noted at one treated skin site at the 48-hour observation.
Two treated skin sites appeared normal throughout the study and the remaining treated skin site appeared normal at the 72-hour observation.

An isolated incident of very slight erythema was noted at one treated skin site at the 48-hour observation.

Two treated skin sites appeared normal throughout the study and the remaining treated skin site appeared normal at the 72-hour observation.

Deviation from protocol: Due to a technician error the animals were decontaminated one hour later than specified in the Protocol. This deviation was considered not to affect the purpose or integrity of the study.

Interpretation of results:

other: EU-GHS criteria not met

Conclusions:

The test material produced a primary irritation index of 0.0 and was classified as NON IRRITANT to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

Executive summary:

Introduction. The study was performed to assess the irritancy potential of the test material GTL waxy raffinate (C15-50; CAS 848301-87-1, EC 482-130-1) to the skin of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

§        OECD Guidelines for the Testing of Chemicals No. 404 “Acute Dermal Irritation/Corrosion” (adopted)

§        Method B4 Acute Toxicity (Skin Irritation) of Commission Directive 2004/73/EC

Results. A single 5-hour, semi-occluded application of the test material to the intact skin of three rabbits produced an isolated incident of very slight erythema at one treated skin site. Two treated skin sites appeared normal throughout the study and the remaining treated skin site appeared normal at the 72-hour observation.

Due to a technician error the animals were decontaminated one hour later than specified in the Protocol. This deviation was considered not to affect the purpose or integrity of the study.

Conclusion. The test material produced a primary irritation index of 0.0 and was classified as non‑irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 31 January 2008 and 08 February 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21/8/2007. Date of signature: 15/10/2007

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Twelve to twenty weeks old.
- Weight at study initiation:2.0 to 3.5 kg
- Housing: The animals were individually housed in suspended cages.
- Diet (e.g. ad libitum): Free access to food (Certified Rabbit Diet) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water was allowed throughout the study.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve limits of 17 to 23°C.
- Humidity (%): The relative humidity was set ti achieve limits of 30 to 70%.
- Air changes (per hr): At least fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continous light and twelve hours darkness.

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 ml of the test material.

Duration of treatment / exposure:

4 h

Observation period:

72 hours

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: A quantity of 0.5 ml of the test material was introduced under a 2.5 cm x 2.5 cm cotton gauze patch and placed in position on the shorn skin.
- Type of wrap if used: The patch was secured in position with a strip of surgical adhesive tape. To prevent the animal interfering with the patch, the trunk of the rabbit was wrapped in an elasticated corset and the animal was returned to its cage for the duration of the 4 hour exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Four hours after application the corset and patch were removed from the animal and any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: Four hours

SCORING SYSTEM:
Approximately one hour following the removal of the patches, and 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored according to the Draize scale (see below):

Other:
Measurement of pH:
The pH of the test material was determined prior to commencement of the study and found to be as follows:
90% v/v aqueous preparation of the test material - pH: approx 8.9 immediately, approx 8.6 after 10 minutes.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

: animal 67060 (female)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Remarks:

: animal 67162 (male)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

mean

Remarks:

: animal 67163 (male)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

: animal 67060 (female)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

: animal 67162 (male)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

: animal 67163 (male)

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No evidence of skin irritation was noted during the study.

Other effects:

No corrosive effects were noted.

Interpretation of results:

other: EU-GHS criteria not met

Conclusions:

The test material produced a primary irritation index of 0.0 and was classified as NON-IRRITANT to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

Executive summary:

Introduction.

The study was performed to assess the irritancy potential of the test material GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0) to the skin of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

• OECD Guidelines for the Testing of Chemicals No. 404 "Acute Dermal Irritation/Corrosion" (adopted 24 April 2002)

• Method B4 Acute Toxicity (Skin Irritation) of Commission Directive 2004/73/EC.

 

Results.

A single 4-hour, semi-occluded application of the test material to the intact skin of three rabbits produced no evidence of skin irritation.

 

Conclusion.

The test material produced a primary irritation index of 0.0 and was classified as non-irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted. The test material did not meet the criteria for classification as irritant according to EU labelling regulations Commission Directive 2001/59/EC (Dangerous Substances Directive) and according to EU CLP regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

2022-02-07 until 2022-07-25

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

26th June 2020

Deviations:

no

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

Specific details on test material used for the study:

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

Strain:

not specified

Details on test animals or tissues and environmental conditions:

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

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”).

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

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

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)

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}

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).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 11 February 2008 and 21 February 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21/8/2007. Date of signature: 15/10/2007

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: Twelve to twenty weeks old.
- Weight at study initiation: 2.0 to 3.5 kg
- Housing: The animals were individually housed in suspended cages.
- Diet (e.g. ad libitum): Free access to food (Certified Rabbit Diet) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water was allowed throughout the study.
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was set to achieve limits of 17 to 23°C.
- Humidity (%): The relative humidity was set to achieve limits of 30 to 70%.
- Air changes (per hr): At least fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continous light and twelve hours darkness.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml

Duration of treatment / exposure:

Single application of test material (72 hours).

Observation period (in vivo):

72 hours.

Number of animals or in vitro replicates:

3

Details on study design:

SCORING SYSTEM:
Immediately after administration of the test material, an assessment of the initial pain reaction was made.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical Draize Scale for Scoring Ocular Irritation.

Any other ocular effects were also noted.

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

: animal 67182

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

: animal 67215

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

: animal 67216

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

: animal 67182

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

: animal 67215

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Remarks:

: animal 67216

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Remarks:

: animal 67182

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Remarks:

: animal 67215

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

mean

Remarks:

: animal 67216

Time point:

24/48/72 h

Score:

0.3

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

: animal 67182

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

: animal 67215

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

: animal 67216

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No corneal or iridial effects were noted.

Minimal conjunctival irritation was noted in all treated eyes one hour after treatment and in two treated eyes at the 24-hour observation.

One treated eye appeared normal at the 24-hour observation and the remaining two treated eyes appeared normal at the 48-hour observation.

Other effects:

Initial pain reaction: Slight initial pain in all animals.

Interpretation of results:

other: EU-GHS criteria not met

Conclusions:

The test material produced the following individual mean scores for opacity, iritis redness and chemosis, as required for classification according to the Globally Harmonised classification Scheme:

Rabbit Number: 67182
Opacity: 0.0
Iritis: 0.0
Conjunctival redness: 0.0
Chemosis: 0.0

Rabbit number: 67215
Opacity: 0.0
Iritis: 0.0
Conjunctival redness: 0.3
Chemosis: 0.0

Rabbit number: 67216
Opacity: 0.0
Iritis: 0.0
Conjunctival redness: 0.3
Chemosis: 0.0

Based on these results the substance is not classified as an eye irritant.

Executive summary:

Introduction.

The study was performed to assess the irritancy potential of the test material GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0) to the eye of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

• OECD Guidelines for the Testing of Chemicals No. 405 "Acute Eye Irritation/Corrosion" (adopted 24 April 2002)

• Method B5 Acute Toxicity (Eye Irritation) of Commission Directive 2004173/EC.

 

Result.

A single application of the test material to the non-irrigated eye of three rabbits produced minimal conjunctival irritation. One treated eye appeared normal at the 24-hour observation and the remaining two treated eyes appeared normal at the 48-hour observation.

The test material produced the following individual mean scores for opacity, iritis redness and chemosis, as required for classification according to the Globally Harmonised classification Scheme:

Rabbit Number 67182

Opacity: 0.0

Iritis: 0.0

Conjunctival redness: 0.0

Chemosis: 0.0

Rabbit number: 67215

Opacity: 0.0

Iritis: 0.0

Conjunctival redness: 0.3

Chemosis: 0.0

Rabbit number: 67216

Opacity: 0.0

Iritis: 0.0

Conjunctival redness: 0.3

Chemosis: 0.0

 

The test material did not meet the criteria for classification as irritant according to Regulation (EC) No 1272/2008.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 November 2007 and 06 December 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: August 2007; Date of certificate: October 2007

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Limited, Bicester, Oxon, UK.
- Age at study initiation: Twelve to twenty weeks old.
- Weight at study initiation: At the start of the study the animals were in the weight range of 2.0 to 3.5 kg.
- Housing: The animals were individually housed in suspended cages.
- Diet (e.g. ad libitum): Free access to mains drinking water and food (Certified Rabbit Diet) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Water (e.g. ad libitum): Free access to mains drinking water and food (Certified Rabbit Diet) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Acclimation period: Acclimatisation period of at least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): The rate of air exchange was at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES: From:0 To:3

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A volume of 0.1 ml of the test material was placed into the conjunctival sac of the right eye
- Concentration (if solution): No data

VEHICLE
- Amount(s) applied (volume or weight with unit):Not applicable
- Concentration (if solution):Not applicable
- Lot/batch no. (if required): Not applicable
- Purity:Not applicable

Duration of treatment / exposure:

72 Hours

Observation period (in vivo):

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment.

Number of animals or in vitro replicates:

Initially, a single rabbit was treated. After consideration of the ocular responses produced in the first treated animal, two additional animals were treated.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): No Data
- Time after start of exposure:No data

SCORING SYSTEM:
The numerical values corresponding to each animal, tissue and observation time were recorded. The data relating to the conjunctivae were designated by the letters A (redness), B (chemosis) and C (discharge), those relating to the iris designated by the letter D and those relating to the cornea by the letters E (degree of opacity) and F (area of cornea involved). For each tissue the score was calculated as follows:
Score for conjunctivae = (A + B + C) x 2
Score for iris = D x 5
Score for cornea = (E x F) x 5

TOOL USED TO ASSESS SCORE: Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Irritant / corrosive response data:

No corneal or iridial effects were noted.
Minimal conjunctival irritation was noted in all treated eyes one hour after treatment and at the 24-hour observation.
All treated eyes appeared normal at the 24-hour observation.

Other effects:

None

IndividualScores and Individual Total Scoresfor Ocular Irritation

Rabbit Number and Sex	66812 Male				66869 Male				66870 Male
	IPR= 2				IPR = 2				IPR = 2
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	1	1	0	0	1	1	0	0	1	1	0	0
B = Chemosis	1	0	0	0	0	0	0	0	1	0	0	0
C = Discharge	1	0	0	0	1	0	0	0	1	0	0	0
Score (A + B + C) x 2	6	2	0	0	4	2	0	0	6	2	0	0
Total Score	6	2	0	0	4	2	0	0	6	2	0	0

 

Individual Total Scores and Group Mean Scoresfor Ocular Irritation

Rabbit Number and Sex	Individual Total Scores At:
	1 Hour	24 Hours	48 Hours	72 Hours
66812 Male	6	2	0	0
66869 Male	4	2	0	0
66870 Male	6	2	0	0
Group Total	16	6	0	0
Group Mean Score	5.3	2.0	0.0	0.0

Draize Scale for Scoring Ocular Irritation

1.	CONJUNCTIVAE
	(A)	Redness(refers to palpebral and bulbar conjunctivae excluding cornea and iris)
		Vessels normal		0
		Vessels definitely injected above normal		1
		More diffuse, deeper crimson red, individual vessels not easily discernible		2
		Diffuse beefy red		3
	(B)	Chemosis
		No swelling		0
		Any swelling above normal (includes nictitating membrane)		1
		Obvious swelling with partial eversion of lids		2
		Swelling with lids about half closed		3
		Swelling with lids half closed to completely closed		4
	(C)	Discharge
		No discharge		0
		Any amount different from normal (does not include small amounts observed in inner canthus of normal animals)		1
		Discharge with moistening of the lids and hairs just adjacent to lids		2
		Discharge with moistening of the lids and hairs a considerable area around the eye		3
	THE TOTAL SCORE = (A + B + C) x 2		MAXIMUM TOTAL = 20
2.	IRIS
	(D)	Values
		Normal		0
		Folds above normal, congestion, swelling, circumcorneal injection (any or all of these or combination of any thereof) iris still reacting to light (sluggish reaction is positive)		1
		No reaction to light, haemorrhage, gross destruction (any or all of these)		2
	THE TOTAL SCORE = D x 5		MAXIMUM TOTAL = 10
3.	CORNEA
	(E)	Degree of Opacity (most dense area used)
		No opacity		0
		Scattered or diffuse areas, details of iris clearly visible		1
		Easily discernible translucent areas, details of iris slightly obscured		2
		Opalescent areas, no details of iris visible, size of pupil barely discernible		3
		Opaque, iris not discernible through the opacity		4
	(F)	Area of Cornea Involved
		One quarter (or less) but not zero		1
		Greater than one quarter but less than half		2
		Greater than half but less than three quarters		3
		Greater than three quarters, up to whole area		4
	THE TOTAL SCORE = (E x F) x 5		MAXIMUM TOTAL = 80
	MAXIMUM TOTAL SCORE POSSIBLE = 110

Interpretation of results:

other: EU-GHS criteria not met

Conclusions:

The test material produced a maximum group mean score of 5.3 and was classified as a MINIMAL IRRITANT (Class 3 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

Executive summary:

Introduction. The study was performed to assess the irritancy potential of the test material GTL waxy raffinate (C15-50; CAS 848301-87-1, EC 482-130-1) to the eye of the New Zealand White rabbit. The method was designed to meet the requirements of the following:

§        OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted)

§        Method B.5 Acute Toxicity (Eye Irritation) of Commission Directive 2004/73/EC

§        United States Environmental Protection Agency Health Effects Test Guidelines OPPTS 870.2400 Acute Eye Irritation August 1998.

Result. A single application of the test material to the non-irrigated eye of three rabbits produced minimal conjunctival irritation. All treated eyes appeared normal at the 48-hour observation.

Conclusion. The test material GTL waxy raffinate (C15-50; CAS 848301-87-1, EC 482-130-1) produced a maximum group mean score of 5.3 and was classified as a minimal irritant (Class 3 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

An in vitro study according to OECD Guideline 439 was performed to assess the skin irritation potential of the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' by means of the Human Skin Model Test.

The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. Also, its intrinsic colour was not intensive and the optical density of the test item in deionised water or isopropanol at 570 nm after blank correction was < 0.08. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed.

Each three tissues of the human skin model EpiDerm^™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes. After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion, thus assuring the quality of the tissues. Treatment with the positive control induced a decrease in the viability compared with the negative control to 4.25%, thus ensuring the validity of the test system. The standard deviations between the three tissue percentage viability values of each group (test item, positive and negative controls) in the main test were below 18 (threshold according to OECD 439: ≤ 18), thus ensuring the validity of the study.

After treatment with the test item the mean viability value was 100.08% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the substance 'Paraffin waxes (Fischer-Tropsch), isomerization' is not considered to possess an irritant potential.

 

Two in vivo studies, both following OECD Guideline 401 and conducted under GLP conditions, were performed to assess the irritancy potential of the closely related substances GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0) and GTL waxy raffinate (C15-50; CAS 848301-87-1, EC 482-130) following a single, 4-hour, semi-occluded application to the intact rabbit skin.

No evidence of skin irritation or corrosion was observed during the studies in three animals. Both test materials produced a primary irritation index of 0.0 and were classified as non-irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

 

Eye irritation

An 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 (OECD 437).

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).

 

Two in vivo studies, both following OECD Guideline 405 and conducted under GLP conditions, were performed to assess the irritancy potential of the closely related substances GTL base oil (C18-50; CAS 848301-69-9, EC 482-220-0) and GTL waxy raffinate (C15-50; CAS 848301-87-1, EC 482-130) following a single application to the rabbit eye.

In both studies, a single application of the test material to the non-irrigated eye of three rabbits produced minimal conjunctival irritation, but all treated eyes appeared normal at the 48-hour observation (at the latest). 

Both test materials did not meet the criteria for classification as irritant according to EU CLP Regulation.

Justification for classification or non-classification

Skin / eye irritation

Based on results of reliable in vitro eye / skin irritation studies on the registration substance itself, and reliable in vivo eye / skin irritation studies on closely related substances, the registration substance 'Paraffin waxes (Fischer-Tropsch), isomerization' does not meet the criteria for classification and labelling for these endpoints (according to EU CLP classification criteria).