4-methoxybenzyl alcohol

Endpoint summary

Description of key information

According to OECD 439, the test item is considered to be irritant to skin. However, the test item is not corrosive to skin as shown in GLP conform study according to OECD 431.

According to OECD 492, the test item is considered to be irritant to eye. However, the test item does not seriously damage the eye as shown in a GLP conform study according to OECD 437.

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:

weight of evidence

Study period:

April 2016 and August 2016

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:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Human Skin Model (EpiDerm Reconstructed Human Epidermis)
- Tissue batch number: 23338
- Delivery date: 24 May 2016
- Date of initiation of testing: 24 May 2016 (pre-incubation phase started)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 minutes exposure) and 37°C(1 hour exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 20 times
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 2.078 +/- 0.070
- Barrier function: ET-50 = 5.64 hrs
- Contamination: sterile

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 (3 min and 60 min exposure)

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL (79.4 µL/cm2)

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µl
- Concentration: 0.8 N

Duration of treatment / exposure:

3 minutes and 60 minutes

Number of replicates:

2 tissues (three wells per tissue)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

#1 (3 minutes exposure)

Value:

95.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

#2 (60 min exposure)

Value:

15.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is considered to be non corrosive to skin under the test conditions chosen.

Executive summary:

The corrosive potential of the test substance was investigated by means of the in vitro Human Skin Model Test with EpiDerm tissues models according to OECD Guideline 431.

The test item was melted in water bath and afterwards added to independent duplicate tissues of EpiDerm for an incubation time of 3 minutes or 60 minutes, respectively. The negative control (deionised water) and the positive control (0.8 N KOH) were tested in parallel. Subsequently, the test and control items were rinsed off the tissues, and a 3 hour incubation period with MTT followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well paltes. The formazan salt was extracted for about 43 hours in the refrigerator. The OD was determined in a microplate reader at 570 nm.

The required acceptability criteria were met. Furhtermore, the test item passed the MTT- and the Colour Interference pre-tests.

The relative absorbance was decreased to 95.4% after 3 minutes exposure to the test item. After an exposure period of 60 the relative absorbance was reduced to 15.7%.

Based on the results, the test item was not considered to be corrosive under the test conditions chosen.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

February 2016 and April 2016

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:

adopted July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation 440/2008, 1st ATP 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: reconstituted human epidermis

Cell type:

non-transformed keratinocytes

Justification for test system used:

The potential of chemical induced skin irritation is usually determined in vivo in the 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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200 SIT kit
- Tissue batch number(s): 23314
- Delivery date: 16 February 2016
- Date of initiation of testing: On day of receipt the pre-incubation phase of the EpiDerm™ tissues started.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35 min at 37°C and 25 min at room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with DPBS at least 15 times; submerged in DPBS at least three times; afterwards once again rinsed with sterile DPBS from the inside and outside
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.683 +/- 0.077
- Barrier function: 6.55 hours
- Contamination: sterile

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin category 2 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg (~ 39 mg/cm2)

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

43.5 hours

Number of replicates:

3 triplicate tissues (3 wells per tissue)

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

12.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3

Value:

10.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

It can be stated that the in this study and under the experimental conditions chosen, the test item is considered to be irritant.

Executive summary:

An in vitro study in accordance with the OECD Guideline OECD 439 was performed to assess the skin irritation potential of the test item.

The test item was melted in a water bath and afterwards 25 mg of the test item was dispensed directly atop each EpiDerm tissue (in total 3) which were wetted with 25 µL DBPS prior application, and spread to match the surface of the tissue. 30 µL of either the negative control (DPBS) or the positive control (5% SLS) were tested in parallel. The incubation time was 60 minutes.

The test item did not reduce MTT, and it did not indicate colour interference. All of the acceptability criteria were met. The absorbance values of the negative control were between 0.8 and 2.8. The positive control induced a sufficient decrease of the relative absorbance as compared to the negative control. The relative standard deviations of three replicates were below 18%.

As a result, the mean relative absorbance was determined to be 11.4% compared to the relative absorbance of the negative control after treatment with the test item. Thereofre, the test item is considered to be irritant to skin under the experimental conditions chosen.

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:

weight of evidence

Study period:

February 2016 and August 2016

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:

July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: isolated bovine cornea

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Characteristics of donor animals: at least 9 months old
- Storage, temperature and transport conditions of ocular tissue: at ambient temperature
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes. The corneae were directly used in the BCOP test on the same day.
- indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3 corneas per group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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. Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring) 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 no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. At the end of the incubation period, the basal opacity was determined (t0). The basal opacity of all corneae was recorded. Each corneae with a value of the basal opacity > 7 was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Yes

POSITIVE CONTROL USED: Yes

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 min

POST-INCUBATION PERIOD: yes (2 hours)

REMOVAL OF TEST SUBSTANCE
- POST-EXPOSURE INCUBATION: 2 hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: OP_KIT opacitometer (changes in the light transmission passing through the cornae)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage according to CLP/EPA/GHS (Cat 1)

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

12.04

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

16.37

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

13.95

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Neither an increase of opacity nor permeability of the cornae could be obsered. However the negative control values were slightly elevated beyond the historical control data of the negative control range (IVIS = 0.560 - 1.70). This elevation is caused by a single cornea, which had a slightly elevated IVIS value. Because the mean IVIS of the three negative control cornea is still below the threshold for categorization (< 3), this deviation is considered irrelevant.
- Acceptance criteria met for positive control: Yes

Interpretation of results:

other: not Category 1 according to GHS criteria

Conclusions:

Based on the results of the OECD 437 study, the test item is not serious eye damaging (Cat. 1) but a prediction for the damage hazard cannot be made.

Executive summary:

To assess the corneal damage potential of the test item, an in vitro study according to OECD TG 437 was investigated. After a first opacity measurement of the fresh bovine cornea (t0), the neat test item, the positive control, and the negative control were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32°C. After the incubation phase the test item, the positive control, and the negative controls were each rinsed from the cornea. Further, teh cornea were incubated for another 120 min at 32°C in a vertical position. Afterwards, opacity was measured a second time (t130). Permeability of the cornea was also determined after opacity measurements.

The acceptance criteria for the negative and positive control were met. Although, the negative values were slightly outside the historical control data range, which is caused by a single cornea and still below the threshold for categorization, this deviation is conidered irrelevant.

In comparison to the negative control, the test item caused an increase of the corneal opacity. The calculated mean in vitro irritancy score was 14.12. In conclusion, the test item is not classified as serious eye damaging but the test item's hazard for eye damaging cannot be predicted.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 2016 and August 2016

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:

July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

June 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EpiOcular tissues

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
This study was performed to assess the eye irritation potential of the test item. In a prevalidation study performed by Avon Products Inc. and MatTek Corporation, the in vitro eye test using the human cornea model EpiOcular™ and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for eye irritancy potential.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular™ tissue consists of normal, human-derived epidermal 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 which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo cornea epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm diameter).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

120 min

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used:
Preparation:
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24- well plate. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. An appropriate volume of EpiOcular™ Assay Medium was warmed to approximately 37 °C. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions. Cultures with air bubbles under the inserts greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (about 17 hours).

Experimental performance
After the overnight incubation, the tissues were pre-wetted with 20 μL of Ca2+ Mg2+ free- DPBS. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 30 minutes. Afterwards, the tissues were incubated with the test and control items. At the end of the exposure time, the test item was removed by extensively rinsing the tissues with Ca2+ Mg2+-free DPBS (brought to room temperature). After the washing steps, the remaining liquid was decanted onto the absorbent material followed by the immediate transfer of the tissues to 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 12 minute immersion temperature (post-soak) at room temperature. Subsequently, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 ml of warm Assay Medium. The tissues were incubated for about 120 minutes at standard culture conditions (post-treatment incubation).

- RhCE tissue construct used, including batch number: EpiOcular™ kits and MTT-100 kits (Lot No. 23707)

- Doses of test chemical and control substances used: 50 µL

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 30 min exposure at 37°C; 12 min post-soak immersion at room temperature; 120 min post-exposure at 37°C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Since the test item did not dye wter or isopropanol and did not directly reduced MTT, additional controls are not required.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device: 560 nm without reference filter using a microplate reader

- Description of the method used to quantify MTT formazan:
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions and rinsed 3 times with DPBS afterwards. Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark. To extract the MTT, the plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken. The extract solution was mixed and two 200 μL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.The OD was determined at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: historical positive and negative control data are provided by the conducting laboratory

- Complete supporting information for the specific RhCE tissue construct used: Certificate of Analysis on test system quality is provided by the supplier

- Positive and negative control means and acceptance ranges based on historical data:
Acceptance range negative control: OD is > 0.8 and < 2.5.
The mean relative viability of the positive control is below 50% of the negative control viability.

- Acceptable variability between tissue replicates for positive and negative controls: < 20%

- Acceptable variability between tissue replicates for the test chemical: < 20%

Irritation parameter:

other: mean rel. absorbance (% of negative control)

Value:

13.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of this OECD 492 study, the test item possesses an eye irritating potential.

Executive summary:

The eye irritation potential of the test item was assessed in an in vitro Human Cornea Model Test according to OECD 492.

Additional tests with freeze-killed or viable tissues were not required as the test item did not prove to be a MTT reducer and did not show colour interference. Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes. All of the acceptability criteria were met. The negative control OD was between 0.8 and 2.5. Tretament with the positive control decreased the relative absorbance below 60% compared with the negative control. The difference of viability between the relating tissues of a single item was less than 20% in the same run (for positive and negative control tissues as well as test item treated tissues).

Irritating effects were observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (13.8%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses an eye irritating potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation study

An in vitro study in accordance with the OECD Guideline OECD 439 was performed to assess the skin irritation potential of the test item.

The test item was melted in a water bath and afterwards 25 mg of the test item was dispensed directly atop each EpiDerm tissue (in total 3) which were wetted with 25 µL DBPS prior application, and spread to match the surface of the tissue. 30 µL of either the negative control (DPBS) or the positive control (5% SLS) were tested in parallel. The incubation time was 60 minutes.

The test item did not reduce MTT, and it did not indicate colour interference. All of the acceptability criteria were met. The absorbance values of the negative control were between 0.8 and 2.8. The positive control induced a sufficient decrease of the relative absorbance as compared to the negative control. The relative standard deviations of three replicates were below 18%.

As a result, the mean relative absorbance was determined to be 11.4% compared to the relative absorbance of the negative control after treatment with the test item. Therefore, the test item is considered to be irritant to skin under the experimental conditions chosen.

 

Skin corrosion study

The corrosive potential of the test substance was investigated by means of the in vitro Human Skin Model Test with EpiDerm tissues models according to OECD Guideline 431.

The test item was melted in water bath and afterwards added to independent duplicate tissues of EpiDerm for an incubation time of 3 minutes or 60 minutes, respectively. The negative control (deionised water) and the positive control (0.8 N KOH) were tested in parallel. Subsequently, the test and control items were rinsed off the tissues, and a 3 hour incubation period with MTT followed. MTT solution was then aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well paltes. The formazan salt was extracted for about 43 hours in the refrigerator. The OD was determined in a microplate reader at 570 nm.

The required acceptability criteria were met.

The relative absorbance was decreased to 95.4% after 3 minutes exposure to the test item. After an exposure period of 60 the relative absorbance was reduced to 15.7%.

Based on the results, the test item was not considered to be corrosive under the test conditions chosen.

 

Supporting studies in animals

Sharp et al. (1978) determined the skin irritation potential of the test substance in a pre-experiment as part of a modified Draize sensitisation in guinea pigs. Four animals were given intradermal injections on shaved with 0.1 mL aliquots of test material (0.25%) and reactions were read after 24 hours. Slight but perceptible irritation without edema was produced at 0.25%. In a second preliminary test, 0.1 mL of the test material (10%) was topical applied to the shaved flank of 4 guinea pigs. Twenty-four hours later reactions were evaluated and a concentration of 10% did not produce irritants effects.

 

In an acute dermal toxicity study by Moreno (1973), 3 -4 rabbits per dose were treated with the test substance at concentrations of 1.25 g/kg, 2.5 g/kg and 5.0 g/kg, respectively. The following effects were observed: at 1.25 g/kg, moderate erythema with slight to moderate edema; at 2.5 g/kg, moderate redness with slight to moderate edema; at 5 g/kg, moderate redness and edema.

 

Draize et al. (1948) investigated the skin irritation potential during an acute and 3 -week subacute skin toxicity study in rabbits. Moderately irritating on repeated application with a primary irritation index of 4. The study has to be considered as disregarded due to missing information on concentration.

 

These results confirm the irritating potential of the test item even if topical application of the test item to guinea pigs did not produce irritant effects. This discrepance of the result of this supporting study may be explained by differences in application and species in comparison to the key study. Furthermore, the test item was used undiluted in the key study. 

 

Supporting studies with humans

In a pre-test for skin sensitization, Kligman (1971) examined the test material at a dose of 5% for its skin irritation potential. The test material was applied to the normal skin of seven subjects under a 48 hour occlusive patch test. No irritation reaction was observed. This indicate that the test item at low concentrations did not produce irritation in humans.

 

Overall Conclusion - Skin irritation/corrosion

Two GLP and Guideline conform in vitro studies are available which are sufficient and reliable to cover the endpoint skin irritation/corrosion. Based on the results, the test item is considered to be irritant to skin.

 

Eye irritation/corrosion study

To assess the corneal damage potential of the test item, an in vitro study according to OECD TG 437 was investigated. After a first opacity measurement of the fresh bovine cornea (t0), the neat test item, the positive control, and the negative control were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32°C. After the incubation phase the test item, the positive control, and the negative controls were each rinsed from the cornea. Further, the cornea were incubated for another 120 min at 32°C in a vertical position. Afterwards, opacity was measured a second time (t130). Permeability of the cornea was also determined after opacity measurements.

The acceptance criteria for the negative and positive control were met. Although, the negative values were slightly outside the historical control data range, which is caused by a single cornea and still below the threshold for categorization, this deviation is conidered irrelevant.

In comparison to the negative control, the test item caused an increase of the corneal opacity. The calculated mean in vitro irritancy score was 14.12. In conclusion, the test item is not classified as serious eye damaging but the test item's hazard for eye damaging cannot be predicted.

 

Eye irritation study

The eye irritation potential of the test item was assessed in an in vitro Human Cornea Model Test according to OECD 492.

Additional tests with freeze-killed or viable tissues were not required as the test item did not prove to be a MTT reducer and did not show colour interference. Each 50 μL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissue for 30 minutes. All of the acceptability criteria were met. The negative control OD was between 0.8 and 2.5. Tretament with the positive control decreased the relative absorbance below 60% compared with the negative control. The difference of viability between the relating tissues of a single item was less than 20% in the same run (for positive and negative control tissues as well as test item treated tissues).

Irritating effects were observed following incubation with the test item. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (13.8%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses an eye irritating potential.

 

Overall Conclusion - Eye irritation/corrosion

Based on the WoE approach consists of OECD TG 437 and OECD TG 492 which are reliable and suitable to fulfill the REACH standard requirements, the test item is considered to be irritating to eye.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on the available experimental data, the substance is considered to be classified for skin irritation cat. 2 (H315) and eye irritation cat. 2 (H319) under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EC) No 2016/918.