3-methyl-1,3-butanediol-1-ethylate

Endpoint summary

Description of key information

Skin corrosion in vitro (reconstructed human epidermis model)

An in vitro skin corrosion study has been conducted with 3-methyl-1,3-butanediol acetate (IPD-AC) in accordance with OECD TG 431 (2016) and in compliance with GLP. The test substance was applied topically to a reconstructed human epidermis model (EpiDerm). Negative (distilled water) and positive (KOH) controls were tested concurrently. The skin tissues were tested in duplicate for 3 and 60 minutes with the test substance or control. The materials were then removed and the tissues incubated for 3 hours. Cell viability was determined by the enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt. The optical density was measured at 570 nm. Viability was determined relative to the negative control tissue. Exposure to IPD-AC for 3 and 60 minutes gave tissue viabilities of 97.7% and 80.7%, respectively.  On the basis of this study, it is concluded that IDP-AC does not require classification for skin corrosivity. 

Skin irritation (reconstructed human epidermis model)

An in vitro skin irritation study has been conducted with IPD-AC in accordance with OECD TG 439 (2015) and in compliance with GLP. The test substance was applied topically to a reconstructed human epidermis model. Negative (Dulbecco’s phosphate buffered saline) and positive (sodium dodecyl sulphate) controls were tested concurrently. The skin tissues were tested in triplicate for 15 minutes with the test substance or control. The materials were then removed and the tissues incubated for 42 hours. Cell viability was determined by the enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt. The optical density was measured at 562 nm. Viability was determined relative to the negative control tissue. The viability of the IDP-AC treated tissues was 111.1%, On the basis of this study, it is concluded that IDP-AC does not require classification for skin irritation. 

Serious eye damage (BCOP)

A bovine corneal opacity and permeability (BCOP) test has been conducted with IPD-AC in accordance with OECD TG 437 (2013) and in compliance with GLP. The test article was applied to ex vivo bovine eyes for 10 minutes. The eyes were incubated for a further 120 minutes. Negative (0.9% w/v sodium chloride) and positive (ethanol) controls were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS for the test substance was >3 and <55 thereby concluding that the test substance does not cause serious damage to eyes, nor can it be not classified for eye irritation.

Eye irritation (EpiOcular)

An EpiOcular test has been conducted with IPD-AC in accordance with OECD TG 492 (2018) and in compliance with GLP. The test substance was applied topically to the EpiOcular tissue for 30 minutes, followed by a 2 hour-post incubation period and immediate determination of cytotoxic effects via the MTT reduction assay, used as an indicator of irritancy through the formation of formazan. Under the conditions of this study the IPD-AC showed irritant effects. The mean relative tissue viability was <60% (12.5%) after a 30 minute exposure (+2 hour post-incubation). Therefore, IPD-AC requires classification. However, this test guideline cannot resolve between CLP Categories 1 and 2.

Eye irritation (STE)

The ability of the test material IPD-AC to cause serious eye damage or serious eye irritation was investigated in a short-term exposure (STE) assay in rabbit cornea SIRC cells, performed to GLP and OECD TG 491.  The mean cell viabilities over the three independent experiments were 76.9% (5% w/w) and 104.3% (0.05% v/v).  The results of this study indicate that IPD-AC does not require classification for eye irritation according to CLP criteria.

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

Adequacy of study:

key study

Study period:

2016-10-08 to 2016-06-08

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:

29 July, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: 161121
Purity: 86.5%
Expiry date: May 2019
Appearance: Clear colourless liquid
Storage conditions: Room temperature in the dark under nitrogen

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not stated

Source strain:

other: Not applicable

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Supplier: MatTek in vitro life science laboratories, Slovakia
- Tissue batch number: 28646
- Assay medium lot number: 081618TMB

ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls. As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
50 µL of the test item were mixed per 300 µL Aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated for 1 hour at 37°C, 5.0% CO2 / 95% air. If the test item was classified as non-corrosive and colouring was detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbed light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential. For quantitative correction of results, the test was performed using two additional living tissues per exposure period treated with 50 µL of the test item (TVT). All steps were then performed exactly as described in the chapter below, except for the MTT-staining of the test item treated tissues, which were incubated in medium without MTT.

EXPERIIMENTAL PROCEDURE
Upon receipt of the EpiDerm, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL pre-warmed assay medium per well. The 6-well plates were pre-incubated in a humidified incubator at 37°C, 5.0% CO2 / 95% air for at least 1 hour. Then the medium was replaced by 0.9 mL fresh assay medium and the surface was dried using a sterile cotton tip. The 6-well plate used for the 3 minute experiment was placed back into the incubator. The other plate was used for the 60 minute treatment. Approximately 1 hour before the end of the first treatment period the MTT solution was prepared and pre-warmed in the incubator.

60 minute experiment: tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval was kept between dosing. Then the 6-well plate was incubated at 37°C, 5.0% CO2 / 95% air.

3 minute experiment: tissues were treated with each dose group in duplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. A constant time interval of 20 seconds was kept between dosing.
After the 3 minute application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed approximately 20 times with PBS (phosphate buffered saline) to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well holding plate containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner.
Then the inserts were dried again and transferred into a prepared 24-well MTT assay plate containing 300 µL pre-warmed MTT solution. The plate was incubated for 3 hours at 37°C, 5.0% CO2 / 95% air.

60 minute experiment: after the 60 minute application, the first insert was removed from the 6-well plate with forceps. Using a wash bottle, the tissue was gently rinsed approximately 20 times with PBS to remove any residual test item. Excess PBS was removed by gently shaking the insert and blotting bottom with blotting paper. The insert was placed in a prepared 24-well holding plate containing 300 µL pre-warmed assay medium per well. All inserts were treated in the same manner.
The inserts were dried again and transferred into a prepared 24-well MTT assay plate containing 300 µL pre-warmed MTT solution. The plate was incubated for 3 hours at 37°C, 5.0% CO2 / 95% air.

3 minute and 60 minute experiment: after the 3 hour MTT incubation period the MTT solution was aspirated. The wells were refilled with PBS and the PBS was aspirated. The rinsing was repeated twice and the tissues were dried. The inserts were transferred into 12-well extraction plates. Two millilitres of isopropanol were pipetted into each skin tissue insert, thus the insert was covered from both sides to extract the complete formazan. The extraction plates were sealed in zip-bags to inhibit isopropanol evaporation. Extraction was carried out over night without shaking at room temperature.
After the extraction period the inserts were pierced with an injection needle to allow the extracts to run through the tissues into the corresponding wells. The inserts were discarded and the extraction plates were placed on a shaker for 15 minutes.
For each tissue 3 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

EVALUATION CRITERIA
Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after 3 minutes and 60 minutes treatment compared to the negative control tissues concurrently treated with Aqua dest (= 100%) according to the following Prediction Model:

STEP 1:
Mean tissue viability (% negative control) < 50% after 3 min exposure = Corrosive

Mean tissue viability (% negative control) => 50% after 3 min exposure AND < 15% after 60 min exposure = Corrosive. A combination of optional Sub-categories 1B and 1C

Mean tissue viability (% negative control) => 50% after 3 min exposure AND =>15% after 60 min exposure = Non corrosive

STEP 2:
Mean tissue viability (% negative control) < 25% after 3 min exposure = Optional Sub-category 1A

Mean tissue viability (% negative control) => 25% after 3 min exposure = A combination of optional Sub-categories 1B and 1C

ASSAY ACCEPTANCE CRITERIA
The assay was considered valid if all the following criteria were met:
- mean absolute OD570 nm of the two negative control tissues of the 3 minute and 60 minute treatment periods was between 0.8 and 2.8,
- mean relative tissue viability of the two positive control tissues of the 60 min treatment period was <15%,
- coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically was =<30%.

QUANTITATIVE MTT ASSESSMENT
If NSCliving was =< 5% relative to the negative control of living epidermis, no correction of the results was necessary.
If NSCliving was > 5% and =< 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected.
If NSCliving was > 30% relative to the negative control of living epidermis, the test item was considered as incompatible

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 µL (79.4 µL/cm2)

Duration of treatment / exposure:

3 and 60 minutes

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

ca. 97.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

ca. 80.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Pre-Experiments

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%.

The mixture of 50 µL test item per 300 µL Aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equalled 0%.

 

Main Experiment:

Results

 

Table 1: Experimental results

Parameter	Negative Control (distilled water)	Test article (IPD-AC)	Positive Control (KOH)
3 minute exposure
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected) ±SD	1.635 ±0.051	1.598 ±0.053	0.160 ±0.018
Mean Relative Tissue Viability [%]	100	97.7	9.8
Coefficient Of Variation [%]***	3.1	3.3	11.0
3 minute exposure
Total Mean OD570 of 2 Replicate Tissues (Blank Corrected) ±SD	1.679 ±0.069	1.355 ±0.036	0.106 ±0.008
Mean Relative Tissue Viability [%]	100	80.7	6.3
Coefficient Of Variation [%]***	4.1	2.7	7.7

 

A summary of the results for both the 3 and 60 minute in vitro skin corrosion experiments are shown above.

 

Mean absolute OD in the negative control group for both treatment periods were deemed acceptable. The mean cell viability in the positive control group in the 60 minute exposure was <15%. The coefficient of variation between the two tissues treated was <30%.

 

Table 2: Test acceptance criteria

	Value	Cut off	Pass/fail
Mean Absolute OD570 nm NK (3 min Experiment)	1.680	0.8= NK =>2.8	Pass
Mean Absolute OD570 nm NK (60 min Experiment)	1.723	0.8=< NK=>2.8	Pass
Mean Relative Tissue Viability [%] of PC (60 min experiment)	6.3	<15%	Pass
CV [%] (in the range of 20 – 100% viability)	2.7 – 4.1	=<30%	Pass

The acceptance criteria were met and the study was accepted as valid.

The potential of the test item to induce skin corrosion was analysed using the three-dimensional human skin model EpiDerm, comprising a reconstructed epidermis with a functional stratum corneum.

 

In the present study 3-Methyl-1,3-butanediol-1-acetate was applied topically to the EpiDerm tissue for 3 and 60 minutes followed by immediate determination of cytotoxic effects via MTT reduction assay.

 

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues.

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equaled 0%.

The mixture of 50 µL test item per 300 µL Aqua dest. and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equaled 0%.

 

The test item exhibited no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary.

 

The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was =50% (97.7%) after the 3 minute treatment and =15% (80.7%) after the 60 minute treatment.

 

The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was >0.8 and =2.8 for each exposure period (1.680, 1.723). The mean relative tissue viability (% negative control) of the positive control was <15% (6.3%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was <30% (2.7% - 4.1%). The negative and positive control treatments met the acceptance criteria (see Table 4), thereby demonstrating the sensitivity and specificity of this assay.

 

It is concluded that 3-Methyl-1,3-butanediol-1-acetate was not corrosive following exposure to a reconstituted three-dimensional human epidermis model (EpiDerm^TM). These conditions included exposure to 3-Methyl-1,3-butanediol-1-acetate for 3 and 60 minutes, with tissue viabilities of 97.7% and 80.7%, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as not corrosive to skin.

Executive summary:

An in vitro skin corrosion study has been conducted with IPD-AC in accordance with OECD test guideline 431 (2016) and in compliance with GLP. The test substance was applied topically to reconstructed human epidermis model. Negative (distilled water) and positive (KOH) controls were tested concurrently. The skin tissues were tested in duplicated for 3 and 60 minutes with the test substance or control. The materials were then removed and the tissues incubated for 3 hours. Cell viability was determined by the enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells). The optical density was measured at 570 nm. Viability was determined relative to the negative control tissue.

It is concluded that 3-Methyl-1,3-butanediol-1-acetate was not corrosive following exposure to a reconstituted three-dimensional human epidermis model (EpiDerm^TM). These conditions included exposure to 3-Methyl-1,3-butanediol-1-acetate for 3 and 60 minutes, with tissue viabilities of 97.7% and 80.7%, respectively.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-08-13 to 2018-08-24

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:

2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: IPDAC-63313
Purity: 96.0%
Expiry date: 30 March 2016
Appearance: Clear colourless liquid
Storage conditions: Room temperature in the dark under nitrogen

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not stated

Source strain:

other: Not applicable

Justification for test system used:

Test system is acceptable in accordance with the OECD test guideline.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Supplier: SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 15-EKIN-049
- Delivery date: 08 December 2015
- Date of initiation of testing: 09 December 2015
- Maintenance medium lot number: 15-MAIN3-054
- Assay medium lot number: 15-ESSC-051

ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls. As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.

PRE-INCUBATION (DAY 0)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert.
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

APPLICATION OF TEST ITEM AND RINSING (DAY 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering 10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of Dulbecco's phosphate buffered saline (DPBS) with calcium and magnesium served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with calcium and magnesium. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for approximately 42 hours.

MTT LOADING/FORMAZAN EXTRACTION (DAY 3)
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

ABSORBANCE/OPTICAL DENSITY MEASUREMENTS (DAY 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

QUANTITATIVE MTT ASSESSMENT
For the test item the relative mean tissue viabilities obtained after the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562 of test item/ mean OD562 of negative control) x 100
Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42-hour post-exposure incubation period. A relative mean tissue viability of =50% is predicted as irritant (category 2). A relative mean tissue viability of >50% is predicted as not irritant.

ASSAY ACCEPTANCE CRITERIA
The assay establishes the acceptance criteria for an acceptable test if the relative mean tissue viability for the positive control treated tissues was =40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is =18%.
The assay establishes the acceptance criteria for an acceptable test if the mean OD562 for the negative control treated tissues was =0.6 and =1.5, and the standard deviation value of the percentage viability is =18%.
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is =18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 µL (26.3 µL/cm2)

Duration of treatment / exposure:

15 minutes

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

ca. 111.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Mean OD562 values and percentage viabilities

 

Substance	Mean OD562	Relative mean viability (%)
Test substance	0.652	111.1
Negative control	0.587	Set at 100%
Positive control	0.055	9.4

The individual and mean OD562values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in the table. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are shown in the table.

The relative mean viability of the test item treated tissues was 111.1% after a 15-minute exposure period and 42-Hour post-exposure incubation period.

It was considered unnecessary to perform IL-1aanalysis as the results of the MTT test were unequivocal.

 

The relative mean tissue viability for the positive control treated tissues was 9.4% relative to the negative control treated tissues and the standard deviation value of the viability was 0.7%. The positive control acceptance criteria were therefore satisfied.

The mean OD562 for the negative control treated tissues was 0.587 and the standard deviation value of the viability was 6.3%. The mean OD562 of the negative control treated tissues was below the acceptance criteria threshold when presented to 3 decimal places but acceptable when rounded to 1 decimal place. As the test item gave an unequivocal non-irritant result, it was considered that the results of this study were acceptable. This is detailed in the report as a deviation. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 2.7%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant.

Executive summary:

An in vitro skin irritation study has been conducted with IPD-AC in accordance with OECD test guideline 439 (2015) and in compliance with GLP. The test substance was applied topically to reconstructed human epidermis model. Negative (Dulbecco’s phosphate buffered saline) and positive (sodium dodecyl sulphate) controls were tested concurrently. The skin tissues were tested in triplicate for 15 minutes with the test substance or control. The materials were then removed and the tissues incubated for 42 hours. Cell viability was determined by the enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells). The optical density was measured at 562 nm. Viability was determined relative to the negative control tissue. The viability of the test article treated tissues was 111.1%, indicating the test article is not irritant to skin in vitro.

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:

weight of evidence

Study period:

14 June 2021 to 3 September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 491 (Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

86.0% purity
IDP-AC
CAS 5205-01-6

Species:

rabbit

Strain:

not specified

Remarks:

Rabbit cornea (SIRC) cells

Details on test animals or tissues and environmental conditions:

Mycoplasma negative rabbit cornea (SIRC) cells specified in the OECD test guideline were obtained from Health Science Research Resources Bank, Japan Health Sciences Foundation. The doubling time was 18-31 hours. Cells were stored frozen in liquid nitrogen, in medium containing 10% FBS and 10% DMSO. The cell passage number was 14-16 for the three experiments.

Vehicle:

physiological saline

Remarks:

5% w/w

Controls:

yes, concurrent no treatment

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

200 microlitres/well
5% (w/w)
0.05% (v/v)

Duration of treatment / exposure:

5 minutes

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

The test material, solvent and positive controls were tested in triplicate.

Details on study design:

- Number of repetitions and replicates used:
The assay was perforemd in triplicate with three independent valud repetitions. An inititial repetition was discounted as it did not meet the acceptance criteria for the positive control.

- Test chemical concentrations used:
5% (w/w), 0.055 (v/v), as specified by the OECD Test Guideline

- Justification for choice of solvent for each test chemical
Based on solubility of the test material and positive control chemicals, and compatability with the test system.

- Duration of exposure to the test chemical:
5 minutes, as specified by the OECD Test Guideline

- Description of any modifications of the test procedure:
None

- Description of evaluation and decision criteria used
Acceptability criteria:
Mean OD in the medium control group >=0.3
Cell viability of the negative control >=80% of the medium control
Cell viability of the positive control 21.1-62.3%
SD for the mean final cell viability <15% for both test material concentrations

Decision criteria:
Category 1 is assigned on the basis of mean cell viability (5% or 0.05%) <=70%
No prediction can be made on the basis of mean cell viability <=70% (5%) and >70% (0.05%)
Category 2 is assigned on the basis of mean cell viability (5% or 0.05%) >70%

- Reference to historical positive control data:
Included in the study report

- Demonstration of proficiency of the laboratory in performing the test method:
Included in the study report

Irritation parameter:

percent tissue viability 

Remarks:

(%)

Run / experiment:

Runs 2-4 (0.05% test material)

Value:

>= 90.4 - <= 114.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

percent tissue viability 

Remarks:

(%)

Run / experiment:

Runs 2-4 (5% test material)

Value:

>= 65.8 - <= 92.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

All acceptance criteria were met for the three independent experiments presented. An initial experiment was rejected.

Summary of results

Group	Experiment #	Viability (%)		Mean (%)	SD (%)
Medium control	2	100.0
	3	100.0
	4	100.0
Negative control	2	97.3	100.0	96.8	5.1
	3	91.5	100.0
	4	101.7	100.0
Positive control	2		38.7	47.2	9.0
	3		56.6
	4		46.3
5%	2		65.8	76.9	13.9
	3		72.5
	4		92.6
0.05%	2		90.4	104.3	12.6
	3		107.8
	4		114.9

 

Interpretation of results:

GHS criteria not met

Remarks:

The results of this study indicate that IPD-AC does not require classification in any GHS Category for eye irritation.

Conclusions:

The results of this study indicate that IPD-AC does not require classification for eye irritation according to CLP criteria.

Executive summary:

The ability of the test material IPD-AC to cause serious eye damage or serious eye irritation was investigated in a short-term exposure (STE) assay in rabbit cornea SIRC cells, performed to GLP and OECD TG 491.  The mean cell viabilities over the three independent experiments were 76.9% (5% w/w) and 104.3% (0.05% v/v).  The results of this study indicate that IPD-AC does not require classification for eye irritation according to CLP criteria.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-10-08 to 2016-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch number: IPDAC-63313
Purity: >96.0%
Expiry date: 30 March 2016
Appearance: Clear colourless liquid
Storage conditions: Room temperature in the dark, under nitrogen

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Adult cattle: 12 to 60 months
Source: Local abattoir as a by product from freshly slaughtered animals
Storage: Hanks' Balanced Salt Solution (HBSS) supplemented with penicillin (100 IU/mL) and streptomycin (100 µg/mL), transported on ice packs to test facility on the same day as slaughter.
Processing: The corneas were prepared immediately on arrival at the test facility.

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL applied to each cornea

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Details on study design:

PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

SELECTION OF CORNEAS AND OPACITY READING
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

TREATMENT OF CORNEAS:
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

PERMEABILITY DETERMINATIONS:
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

HISTOPATHOLOGY:
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

SCORING SYSTEM:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

The condition of the cornea was visually assessed post treatment and post incubation.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

41.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Does not cause serious eye damage or meet the criteria of no classification for irritation.

 

Treatment	In vitro Irritancy Score
Test substance	41.7
Negative control	1.0
Positive control	55.9

 

The positive control In Vitro Irritancy Score fell slightly short of the range of 29.6 to 52.0. However, as this control is intended to show the sensitivity of the test and the scores were only marginally higher than the acceptance range it was considered that the function of the positive control group was not impaired. The positive control acceptance criterion was not satisfied and this is described as a deviation in the report. The negative control gave opacity of =<2.9 and permeability of =<0.103. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:

study cannot be used for classification

Conclusions:

No prediction of eye irritation can be made.

Executive summary:

A bovine corneal opacity and permeability test has been conducted with IPD-AC in accordance with OECD test guideline 437 (2013) and in compliance with GLP. The test substance was applied to ex vivo eyes for 10 minutes. The eyes were incubated for 120 minutes. Negative (0.9% w/v sodium chloride) and positive (ethanol) controls were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS for the test substance was >3 and =< 55 thereby concluding that the test substance does not cause serious damage to eyes, nor can it be not classified for eye irritation. No prediction of eye irritation can be made.

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-10-10 to 2018-10-11

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:

2018

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

3-methyl-1,3-butanediol-1-acetate
CAS number: 5205-01-6
Batch number: 161121
Appearance: Colourless liquid
Storage conditions: Room temperature
Purity: 86.5%
Expiry date: May 2019

Species:

other: EpiOcular (reconstructed human cornea-like epithelium)

Strain:

other: Supplied by MatTex Corporation

Details on test animals or tissues and environmental conditions:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Supplier: MatTek in vitro life science laboratories, Slovakia
- Tissue batch number: 27073
- Cell source: Keratinocytes, strain: 4F1188

Cell culture media:
- Assay medium (MatTex Corporation)
- Lot no.: 100818ISA
- Vehicle / postive controls: distilled water / methyl acetate (MatTex Corporation)

ASSESSMENT OF DIRECT TEST ITEM REDUCTION OF MTT
To check the non-specific MTT-reducing capability of the test item 50 µL of the test item was mixed per 1 mL MTT medium and incubated for 3 h in a humidified incubator at 37°C, 5.0% CO2 / 95% air. If the mixture turned blue/purple, the test item was presumed to have reduced MTT. The part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues if the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value. For quantitative correction of results, two killed tissues were treated with 50 µL of the test item (KT) and one tissue was treated with 50 µL of the negative control (Aqua dest.; KU), respectively.

If the viability difference of the two identically treated killed test item treated tissues (KT) was > 20% the killed control was considered as non-qualified.

If NSMTT was =< 60% relative to the negative control of living tissues it was used for quantitative correction of the results by calculation of the killed control corrected viability (KCCV) which was considered for classification of the test item.

If NSMTT was > 60% relative to the negative control of living tissues the results obtained should be taken with caution as this is the cut-off used to distinguish classified from not classified test items.

If uncorrected ODKT of the tissue extracts fell outside the linear range of the spectrophotometer the test item was considered as incompatible with the test method.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
To check the colouring potential of the test item 50 µL of the test item ws mixed per 1 mL Aqua dest. and per 2 mL isopropanol each in a 6-well plate. The water solution was incubated for at least 1 h in a humidified incubator at 37°C, 5.0% CO2 / 95% air. The isopropanol solution was shaken on a plate shaker for 2 to 3 h. After the respective incubation period, 2 x 200 µL aliquots per test solution were transferred into a 96-well plate, using 200 µL Aqua dest. and isopropanol as respective blanks and OD was measured in a range of 570 ± 30 nm without reference wavelength in a plate spectrophotometer. If one of the two ODnet was > 0.08, or if the intrinsic colour of the test item is blue, black or dark-purple, the test item was checked for its tissue-colouring potential. For quantitative correction of results, the test was performed using two additional living tissues treated with 50 µL of the test item (TVT) if the mean relative tissue viability of the test item treated tissues (TM) was above the 60% threshold value.

If the viability difference of the two identically treated additional viable test item treated tissues (TVT) was > 20% the colour control was considered as non-qualified.

If NSCliving was =< 60% relative to the negative control of living tissues the mean relative tissue viability of the test item treated tissues (TM) was corrected to the NSC-corrected mean relative tissue viability (NSCCV) which was considered for classification of the test item.

If NSCliving was > 60% relative to the negative control of living tissues the results obtained should be taken with caution as this is the cut-off used to distinguish classified from not classified test items.

If uncorrected ODTVT of the tissue extracts fell outside the linear range of the spectrophotometer the test item was considered as incompatible with the test method.

For test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 50 µL of the test item (TKT). All steps were performed exactly as described in the chapter below, except for the MTT-staining of the test item treated with tissues, which were incubated in medium without MTT. The non-specific colour of additional killed tissues (NSCkilled) was then calculated.

The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.

EXPERIIMENTAL PROCEDURE
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h in a humidified incubator at 37°C, 5.0% CO2 / 95% air. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37°C, 5.0% CO2 / 95% air for 16 - 24 h.

After the overnight incubation the tissues were pre-treated with 20 µL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37°C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye.

Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. Then the 6-well plate(s) were incubated for 30 min at 37°C, 5.0% CO2 / 95% air. At the end of the exposure period, the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing, the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 12 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 120 min at 37°C, 5.0% CO2 / 95% air.

After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h at 37°C, 5.0% CO2 / 95% air.

After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 24-well “extraction plates“, containing 2 mL of isopropanol. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out after storage overnight in the dark at 2 - 8 °C. At the end of the extraction period the tissues were pierced and the liquid within each insert was decanted into the well from which it was taken.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.

For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied (volume): 50 µL (83.3 µL/cm2)
- Concentration (if solution): dosed as recieved (undiluted)

VEHICLE
- Amount applied (volume): 50 µL
- Concentration (if solution): n/a
- Lot/batch no. (if required): RNBG3520
- Purity: sterile

Duration of treatment / exposure:

30 minutes / application to tissue surface

Observation period (in vivo):

n/a in vitro method

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

2 in vitro replicates/treatment group

Details on study design:

- Pre-incubation:
One 6-well plate was prepared for each treatment, 2 culture inserts and the medium (1.0 mL) was added to each well of the plate and pre-incubated for 60 minutes in a CO2 incubator. After 60 minutes of pre-incubation the plates were taken out of the CO2 incubator and culture inserts were transferred to the lower wells of the 6-well plates. The plates were incubated for 18-19 h in CO2 incubator.

- Exposure to the test material and rinsing:
After pre-incubation tissues were treated with each dose group in duplicate, starting with the negative control. The test article (50 µL) was added to the surface of the tissues. When the test materials were not spread over the entire tissue surface, the culture inserts were gently tapped to penetrate the test material into the entire tissue. The exposure time was 30 minutes in a CO2 incubator.

At the end of the exposure period tissues were washed with PBS to remove any residual test article. Excess PBS was removed by blotting bottom with blotting paper. After completion of rinsing, the culture inserts were promptly transferred to a 12 well plate containing medium and left to stand for 12 minutes. At the end of the 12 minute period, the inserts were transferred to a 6-well plate containing medium and incubated for 2 h in a CO2 incubator.

Cytotoxicity analysis (MTT):
Following the post-incubation period, the inserts were transferred in a prepared 24-well plate containing 300 µL pre-warmed MTT medium (1 mg/mL) and further incubated for 3 h, under the same conditions as previously stated.

After the 3 h MTT incubation period, the inserts were transferred to a 24-well plate containing isopropanol (2 mL/well) in order to extract the formazan. Extraction was carried out protected from light at room temperature for 2 hours.

The extract were transferred into a 96-well plate and the optical density was measured at 570 nm without reference wavelength in a plate spectrophotometer.

Irritation parameter:

in vitro irritation score

Value:

ca. 12.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

The mean absolute OD570 nm of the negative control fell within the cut off range (> 0.8 and < 2.5). The ean relative tissue viability of the positive control was <50% confirming the validity and specificity of the assay to detect ocular irritants. The difference in viability of each dose group was <20%. The assay was therefore considered acceptable and valid (refer to Table 1)

Test Acceptance Criteria and Validity

 

Table 1: Test acceptance criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nm VC	1.866	0.8 < NK < 2.5	pass
Mean Relative Viability PC [%]	27.6	< 50%	pass
Max. Difference of % Viability [%]	8.5	< 20%	pass

VC: vehicle control

PC: positive control

 

Table 2: historical control data (2017 – 2018)

	Absolute OD570 nm VC	Relative Viability PC [%]	Difference of Viability [%]
Mean ±SD	1.691 ±0.275	23.7 ±13.2	7.0 ±7.6
Range of LCL - UCL	1.142 – 2.240	0.0 – 50.0	0.0 – 22.2
n	40	40	173

LCL: Lower control limit (95%, mean – 2*SD)

UCL: Upper control limit (95%, mean + 2*SD)

n: number of control values

VC: vehicle control

PC: positive control

 

Results:

Pre-Experiments

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

 

The mixture of 50 µL test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSCliving equalled 0%.

 

Experiment main:

The potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium.

 

In the present study 3-Methyl-1,3-butanediol-1-acetate was applied topically to the EpiOcular tissue for 30 min followed by 12 min post-soaking incubation after removal of the test item. After a 120 min post-treatment period cytotoxic effects were determined via MTT reduction assay.

 

Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with Aqua dest.

 

The mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

 

The mixture of 50 µL test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSCliving equalled 0%.

 

The test item showed irritant effects. The mean relative tissue viability (% negative control) was = 60% (12.5%).

 

The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.866). The mean relative tissue viability (% negative control) of the positive control was < 50% (27.6%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (8.5%).

 

Table 3: Result of the Test Item 3-Methyl-1,3-butanediol-1-acetate

	Negative Control	Positive Control	Test Item
Mean Absolute OD570	1.866****	0.545	0.270
Total Mean OD570 of the 2 Replicate Tissues (Blank Corrected) ±SD	1.824* ±0.109	0.502 ±0.107	0.228 ±0.023
Relative Tissue Viability Difference [%]***	8.5	8.3	1.8
Mean Relative Tissue Viability [%]	100.0	27.6**	12.5

* Corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability

** Mean relative tissue viability of the positive control is < 50%

*** Relative tissue viability difference of replicate tissues is < 20%

**** The mean absolute OD570 of the negative control is = 0.8 and = 2.5

Interpretation of results:

other: irritant, but cannot resolve between GHS Categories 1 and 2

Conclusions:

Under the conditions of this study the IPD-AC showed irritant effects. The mean relative tissue viability was <60% (12.5%) after a 30 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, IPD-AC requires classification and labelling. However, this test guideline cannot resolve between GHS Categories 1 and 2.

Executive summary:

In the present study the eye irritating potential of 3-Methyl-1,3-butanediol-1-acetate (IPD-AC) was examined. The test substance was applied topically to the EpiOcular™ tissue for 30 minutes, followed by a 2 hour post incubation period and immediate determination of cytotoxic effects via the MTT reduction assay, used as an indicator of irritancy through the formation of formazan. The test was conducted in accordance with GLP and OECD test guideline 492.

 

Pre-experimental work confirmed that the mixture of 50 µL test item per 1 mL MTT medium showed no reduction of MTT as compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.

 

The mixture of 50 µL test item per 1 mL Aqua dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSCliving equalled 0%.

 

Irritant potential of the test article was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with distilled water. The positive control, methyl acetate viability was <50%, thereby confirming that irritants could be detected in this test system.

 

The test item showed irritant effects. The mean relative tissue viability (% vehicle control) was <60% (12.5%) after 30 minute treatment and 2 hour post incubation.

 

The mean absolute OD570 nm of the negative control fell within the cut off range (> 0.8 and < 2.5). The mean relative tissue viability of the positive control was <50% confirming the validity and specificity of the assay to detect ocular irritants. The difference in viability of each dose group was <20%. The assay was therefore considered acceptable and valid.

 

Under the conditions of this study the IPD-AC showed irritant effects. The mean relative tissue viability was <60% (12.5%) after a 30 minute exposure (+ 2 hour post-incubation). Therefore, according to Annex I for Regulation (EC) 1272/2008 the active ingredient, IPD-AC requires classification and labelling. However, this test guideline cannot resolve between GHS Categories 1 and 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No studies are available. IPD-AC is not classified for skin corrosivity, therefore there is no need to considered further classification for potential respiratory tract irritation (e.g. EUH071).

Justification for classification or non-classification

Comparison with the CLP criteria

Skin corrosion and skin irritation

IPD-AC was investigated in a study of skin corrosion potential using a reconstructed human epidermis model in vitro, conducted according to OECD TG 431.  Based on the criteria specified in the test guideline, IPD-AC does not require classification for skin corrosion (CLP Category 1).

IPD-AC was investigated in a study of skin irritation potential using a reconstructed human epidermis model in vitro, conducted according to OECD TG 439.  Based on the criteria specified in the test guideline, IPD-AC does not require classification for skin irritation (Category 2).

Consequently, based on the available data, IPD-AC does not require classification for skin corrosion (CLP Category 1) or skin irritation (CLP Category 2). 

Serious eye damage and eye irritation

IDP-AC was investigated in a BCOP in vitro study conducted according to OECD TG 437.  Based on the evaluation criteria specified in the test guideline, a test material can be assigned to CLP Category 1 (serious eye damage), or can be identified as not requiring classification in either Category.  No prediction can be made for test materials not meeting these criteria.  The results of the study with IDP-AC did not meet either the criteria for classification in Category 1, or the criteria for no classification.  Consequently, no standalone prediction for the classification of IPD-AC can be made on the basis of this study.

IPD-AC was investigated in the EpiOcular in vitro irritation model conducted according to OECD TG 492.  Based on the evaluation criteria specified in the test guideline, a test material can be identified as not requiring classification in either Category.  No prediction can be made for test materials not meeting these criteria.  The results of the study with IDP-AC did not meet the criteria for no classification.  Consequently, no prediction for the classification of IPD-AC can be made on the basis of this study.  The OECD guideline notes that, in the case of test material for which no prediction can be made, further testing with other test methods will be required.  This is because the test method shows a certain number of false positive results, and cannot resolve between Category 1 and 2.

IPD-AC was also investigated in the STE in vitro irritation model conducted according to OECD TG 491.  Based on the evaluation criteria specified in the test guideline, a test material can be identified as not requiring classification in either Category.  No prediction can be made for test materials not meeting these criteria.  The results of the study with IDP-AC met the criteria for no classification.

According to ECHA REACH guidance (Chapter R.7a), results for eye irritation from in vitro tests not permitting a clear decision on classification should be clarified by performing a definitive in vivo study.  As in vivo testing is not permitted at Annex VII, The REACH Guidance specifies a weight of evidence approach in order to conclude on the eye irritation hazard profile.  Where the data indicate eye irritation potential but are not sufficient to distinguish between classification categories, precautionary classification in Category 1 is specified.  Where data are inconsistent, the Guidance states that a weight of evidence approach may support classification in Category 2.

A classification decision for eye irritation can therefore be made on the basis of the available data, including studies of skin corrosion and irritation.  IPD-AC is not classified for skin corrosion (Category 1), therefore classification for serious eye damage (Category 1) does not apply by default.  IDP-AC is not classified for skin irritation (Category 2).  No prediction for eye irritation classification can be made on the basis of the BCOP or EpiOcular studies.  Both of these studies are able to identify substances not requiring classification in either category for eye irritation, but the criterion were not met for IPD-AC.  In contrast, the STE assay indicates that IPD-AC does not require classification in either category for eye irritation.  Taking into account inconsistencies in the data and following a precautionary approach, IPD-AC is classified in Category 2 for eye irritation.

Respiratory tract:

No studies are available.  The available data from skin and eye irritation do not indicate any potential for respiratory tract irritation.

Overall conclusion

IPD-AC is not classified for skin corrosion or irritation on the basis of the available data

IPD-AC is classified in Category 2 (Causes serious eye irritation) using the weight of evidence and following a precautionary approach in line with ECHA REACH Guidance

IPD-AC is not classified for respiratory tract corrosion or irritation in the absence of any relevant data