Reaction mass of [2,2,4-trimethyl-1-(2-methylpropanoyloxy)pentan-3-yl] benzoate and [2,2,4-trimethyl-3-(2-methylpropanoyloxy)pentyl] 2-methylpropanoate and (3-benzoyloxy-2,2,4-trimethylpentyl) benzoate

Endpoint summary

Description of key information

Skin irritation

OECD439(EpiSkin): not irritant

Eye irritation

OECD437 (BCOP): not corrosive

OECD492 (EIT): not irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27.06.2018-01.10.2018

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)

GLP compliance:

yes

Specific details on test material used for the study:

Batch (Lot) Number: V046812101
Expiry date: Clear colourless liquid
Storage Conditions: At room temperature
Test Facility test item number: 209637/A
Test item handling: No specific handling conditions required

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model TM (SkinEthic Laboratories, Lyon, France).
- Tissue batch number(s): EPISKIN-SMTM, 0.38 cm2, Batch no.: 18 EKIN 035
- Production date: August 28, 2018
- Date of initiation of testing: August 28, 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 ml MTT solution (0.3 mg/ml in PBS)
- Incubation time: 3h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μl
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5% SDS in PBS

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μl
- Concentration (if solution): 100% (undiluted)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl
- Concentration (if solution): 5% SDS in PBS

Duration of treatment / exposure:

15 ± 0.5 minutes

Observation period:

42 hours

Number of animals:

not used, in vitro study

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the tissues were washed with phosphate buffered saline to remove residual test item
- Time after start of exposure: 15 ± 0.5 minutes

OBSERVATION TIME POINTS
The skin tissues were incubated for 42 hours at 37°C. After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 µL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69 hours.

SCORING SYSTEM:

A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Data interpretation of test items

Relative mean viability of 3 individual tissues after 15 minutes of exposure and 42 hours of post incubation Prediction to be considered

≤ 50% of the mean viability of the negative controls Category 1 or Category 2
(additional information on corrosion needed)

> 50% of the mean viability of the negative controls No category

Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

Irritation / corrosion parameter:

% tissue viability

Value:

105

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, at least 25 μl of the test item was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed. No color change were observed.
- Colour interference with MTT: The test item was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the color interference, 10 μl of the test item was added to 90 μl Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μl Milli-Q water was tested concurrently. At the end of the shaking period a color check was performed. No color changes were observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 105%. The absolute mean OD570(optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range.
- Acceptance criteria met for positive control: the positive control had a mean cell viability of 26% after 15 ± 0.5 minutes exposure. The mean relative tissue viability of the positive control should be <=40% relative to the negative control.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 8%. The SD calculated from individual % tissue viabilities of the three identically treated replicates should be <=18.

Mean Absorption in the In Vitro Skin Irritation Test

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	1.012	1.109	1.170	1.097	±	0.079
Test substance	1.218	1.168	1.082	1.156	±	0.069
Positive control	0.288	0.327	0.253	0.290	±	0.037

OD = optical density

SD = Standard deviation

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.0422). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in the In VitroSkin Irritation Test

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	7.2
Test substance	105	6.3
Positive control	26	3.4

	Negative control (absorption; OD570)	Positive control (absorption; OD570)
Range	0.422 – 1.547	0.023 – 0.437
Mean	0.98	0.13
SD	0.18	0.08
n	174	173

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of November 2014 to November 2017.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate is non-irritant in the in vitro skin irritation test under the experimental conditions described and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The objective of this study was to evaluate the test substance for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item was applied undiluted (25 µl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive control had a mean cell viability of 26% after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 105%. Since the individual values were above 50% test item is considered to be non-irritant.

In conclusion, the test substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report andshould not be classified.

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:

22 March 2018 - 14 May 2018

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)

GLP compliance:

yes

Specific details on test material used for the study:

- Source and lot/batch No.of test material: V046803801
- Purity test date: 22 February 2018
- Expiration date of the lot/batch: 06 February 2019
- Storage condition of test material: room temperature, in the dark

Species:

other: in vitro study

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Protocol: Based on the expected high eye irritation potential of the test article, the study was conducted according to the Top-Down Approach; therefore, testing began with the Bovine Corneal Opacity and Permeability test (BCOP). Five corneas were treated with 0.75 ml of test substance EC 911-926-8. Opacity measurements and sodium fluorescein permeability were determined.
- Source: The bovine eyes were from received from Spear Products on 22 Mar 2018. The eyes were transported to MB Research on wet ice in a container containing Hanks’ Balanced Salt Solution (HBSS) with penicillinstreptomycin.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32±1°C
- Cell Culture Conditions: The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential
Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine
(Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 ul
- The test item was tested neat.

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minute

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Corneas from eyes that were free of visible defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS. The dissected corneas were mounted in specially designed holders that are separated into anterior and posterior chambers that are filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects.
The entire holder with the corneas was incubated at 32±1°C and allowed to equilibrate for at least one hour, but not longer than two hours. Following the equilibration, the holders containing the corneas were
removed from the incubator. The MEM solution was removed from both chambers and the chambers refilled with fresh MEM solution. At this time, five corneas were selected for dosing with the test article and two were selected as controls.

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were examined and any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded.
A pre-exposure determination of opacity was made for each of the two control corneas by measuring each against the two blanks supplied by the OP-KIT opacitometer (Electro-Design Corporation, RIOM, France). A pre-exposure determination of opacity was also made for each of the five corneas designated for the test article treatment group by measuring against each control cornea (a total of 10 determinations per test article treatment. Any cornea with an opacity value greater than 7 units was discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
yes

SOLVENT CONTROL USED (if applicable)
no

POSITIVE CONTROL USED
yes

APPLICATION DOSE AND EXPOSURE TIME
750ul 10 min +/- 1min

TREATMENT METHOD:
closed chamber

POST-INCUBATION PERIOD:
yes 120 min

REMOVAL OF TEST SUBSTANCE
Number of washing steps after exposure period: 2

- POST-EXPOSURE INCUBATION:
the corneas were incubated for 120 +/- 10 minutes at 32 +/- 1 degC.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of a 0.4% sodium fluorescein solution in Dulbecco's phosphate buffered saline (DPBS). A Spectronic 20-D Colorimeter Spectrophotometer (Milton Roy, model: 333175) was calibrated prior to use on the same day of dosing. After 90±5 minutes, the fluid from the posterior chamber of each corneal holder was removed and the amount of dye that passed through the cornea (permeability) was measured as the optical density at 490nm (i.e., the OD490nm).
Plate Reader Linearity Check
The linearity of the plate reader used for optical density (OD) determination was verified prior to its use inthe EIT assay. A dilution series of trypan blue was prepared and 200 μl aliquots were pipetted into a 96-well plate. The optical density of the plate wells was measured at a wavelength of 570 nm (OD570), with no reference wavelength. A regression line and an R-squared value were generated using Microsoft Excel® 2007. Verification is considered acceptable if the R-squared value is greater than 0.999.
Test Article Reduction of MTT
A volume of 50 μl of the test article was mixed with 1 ml of MTT solution (1 mg/ml methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium [DMEM]). A negative control (50 μl of tissue culture water, TCH2O) was tested concurrently. The solutions were incubated in the dark at 37±1°C, 5±1% CO2 for 3 hours in a six-well plate. After incubation, the solutions were visually inspected for purple coloration, which indicates that the test article reduced MTT. Since tissue viability is based on MTT reduction, direct reduction by a test article can exaggerate viability, making a test article seem less irritating than its actual irritation potential. The test article did not reduce MTT and the assay continued as per the protocol.
Assessment of Coloring or Staining Materials
Since the test article was non-colored, it was assessed to determine if it would become colored in water or extractant. A volume of 50 μl of the test article were incubated in a six-well plate with 1 ml of tissue culture water for at least one hour in a humidified 37±1°C, 5±1% CO2 incubator. An additional 50 μl of the test article were added to 2 ml of extractant (isopropanol) and incubated for 2 to 3 hours in a six-well plate, at room temperature with shaking. Two 200-μl aliquots of the test article plus TCH2O or test article plus extractant from each well were transferred to a 96-well plate and measured at 570 nm using a plate reader (μQuant Plate Reader, Bio-Tek Instruments, Winooski, VT). No color developed in the water or the extractant, resulting in an OD570 no more than 0.08 after subtraction of a blank (TCH2O or isopropanol, respectively), so no colorant controls were added to the assay.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The corrected mean opacity score was calculated using the control and treated cornea two-hour opacity values as determined from the opacitometer. The 10-minute scores were not used for the final In Vitro Irritation Score (IVIS) calculations as they may be indicative of early reversible effects not associated with the ultimate ocular potential. The corrected mean optical density score was calculated using the control and treated optical density values from the fluorescein permeability analysis. The IVIS of the test article and positive control were calculated as follows:

IVIS = Corrected Mean Opacity Score + (15 x Corrected Mean Optical Density Score)

The IVIS of the negative control article was calculated by adding the mean two-hour opacity score to fifteen times the negative control mean optical density score.

IVIS = Mean Opacity Score + (15 x Mean Optical Density Score)

The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1

DECISION CRITERIA:
The assay is considered acceptable if:
 The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.

Irritation parameter:

in vitro irritation score

Value:

< 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0,17

Positive controls validity:

valid

Remarks:

30,51

Remarks on result:

no indication of irritation

Remarks:

-0,29

Irritation parameter:

cornea opacity score

Value:

< 0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0,0

Positive controls validity:

valid

Remarks:

21,0

Remarks on result:

no indication of irritation

Remarks:

-0,33

Irritation parameter:

fluorescein leakage

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0,011

Positive controls validity:

valid

Remarks:

0,634

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes, The ethanol positive control IVIS was 30.51, which fell within the acceptance range of 16.64 to 38.80 (± 2 standard deviations of the historical mean of 27.72).

Interpretation of results:

study cannot be used for classification

Conclusions:

The BCOP IVIS was -0.29, the corrected mean opacity score was -0.33, and the corrected mean optical density (permeability) score was 0.003. Because the IVIS was less than 55, this result indicated that the test article did not cause serious eye damage, thereby ruling out GHS Category 1.

Executive summary:

Based on the expected high eye irritation potential of the test article, the top-down approach was taken.The objective of this first study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The mean in vitro irritancy score of the positive control (Ethanol) was 30,5 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 

The test item was tested neat. The test item did not induce ocular irritation, resulting in a mean in vitro irritancy score of -0,29 after 10 minutes of treatment. The calculated In Vitro Irritancy Score was less than 55, indicating that the test article did not cause serious eye damage, and thereby ruling out GHS Category 1. In order to reach a definitive result for acute eye hazard classification , the EpiOcular™ Eye Irritation Test was performed

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 March 2018 - 14 May 2018

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)

GLP compliance:

yes

Specific details on test material used for the study:

- Source and lot/batch No.of test material: V046803801
- Purity test date: 22 February 2018
- Expiration date of the lot/batch: 06 February 2019
- Storage condition of test material: room temperature, in the dark

Species:

other: in vitro study

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Protocol: In order to reach a definitive result for acute eye hazard classification, the EpiOcular™ Eye Irritation Test (EIT) was performed. MatTek EpiOcular™ tissue samples were treated in duplicate with the test article, negative control and positive control for 30 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using thiazolyl blue tetrazolium bromide (MTT) uptake and reduction. The absorbance of each sample was measured at 570 nm. The viability was then expressed as a percent of negative control values.
- Source: MatTek Corporation
- Cell line: The EpiOcular™ human cell construct
- Lot: 27424
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37±1°C
- Cell Culture Conditions: a humidified atmosphere of 5±1% CO2 in air

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μl

Duration of treatment / exposure:

30 ± 2 minutes

Observation period (in vivo):

120 ± 15 minutes

Duration of post- treatment incubation (in vitro):

12 minutes

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
The EpiOcularTM tissues Lot 27424 kit F, were received from Mat-Tek on 10 Apr 2018 and refridgerated at 2-8 deg C. Cultures were pre-incubated at 37±1°C in a humidified atmosphere (80-100%) containing 5±0.5% CO2 for 60 min. At the end of the first pre-incubation period, the medium was replaced by 1 mL of fresh assay medium. Further, the tissues were pre-incubated 20 ± 4 hours at 37°C. No correction was made for the purity/composition of the test item. After pre-incubation tissues were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS, then 50 μL of control and test items were applied topically onto the tissue surface and incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were used per treatment, negative and positive controls. After exposure period, each tissue was rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove any residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 ml of previously warmed Assay Medium (room temperature) for a 12 min immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were transferred to warm Assay Medium and were incubated for 120±15 minutes at 37°C.

- RhCE tissue construct used, including batch number
EpiOcular™ human cell construct Lot: 27324

- Doses of test chemical and control substances used
50 μL

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
Exposure: 30 ± 2 minutes at 37.0 ± 1.0°C
Post-exposure immersion: 12 ± 2 minute at room temperature
Post-exposure incubation: 120±15 minutes at 37°C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μl of the test item was added to 1 ml MTT solution (1 mg/ml MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 μl tissue culture water was tested concurrently. If the MTT solution color turned blue / purple or if a blue/purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay. The test item did not reduce MTT.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
2

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
uQuant Plate Reader, Bio-Tek Instruments, Winooski, VT; 570nm

- Positive and negative control means and acceptance ranges based on historical data
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be >0.8 and <2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.

- Acceptable variability between tissue replicates for positive and negative controls
- Acceptable variability between tissue replicates for the test chemical
The SD calculated from individual % tissue viabilities of the two identically treated replicates should be <20%.

Irritation parameter:

in vitro irritation score

Run / experiment:

The mean tissue viability (%)

Value:

94.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not observed

ACCEPTANCE OF RESULTS:
The mean OD570 of the negative control tissues was 1.986 (which met the acceptance criterion of greater than 0.8 and less than 2.5). The mean relative viability of positive control tissues was 31.2%, which met the acceptance criterion of less than 50%. The differences in viability between identically treated tissues were 0.17% to 7.63%, which met the acceptance criterion of less than 20%. All controls passed the acceptance criteria.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate was not irritating to the eye in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as not requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

In order to reach a definitive result for acute eye hazard classification, the EpiOcular™ Eye Irritation Test (EIT) was performed. MatTek EpiOcular™ tissue samples were treated in duplicate with the test article, negative control and positive control for 30 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 31.2% after 30+-2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of two tissues treated with the test item was less than 8%, indicating that the test system functioned properly. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30+-2 minutes treatment with the test item compared to the negative control tissues was 94.3%. Since the mean relative tissue viability for the test item was greater than 60% after 30 +- 2 minutes treatment it is considered to be non-irritant to the eye. In conclusion. The test item is identified as not requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate

was investigated for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM^TM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes. The test item was applied undiluted (25 µl), directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive control had a mean cell viability of 26% after 15 ± 0.5 minutes exposure. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 105%. Since the individual values were above 50% test item is considered to be non-irritant.

In conclusion, test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report andshould not be classified.

Eye irritation

BCOP

The test item was suspected to be severely irritating or corrosive to the eye; therefore, the top-down approach was followed and the testing began with the BCOP test. The BCOP test is used for discriminating Category 1 effects (serious eye damage) from GHS No Category.

The objective of this first study was to evaluate the eye hazard potential of the test item as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The mean in vitro irritancy score of the positive control (Ethanol) was 30,5 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. 

The test item was tested neat. The test item did not induce ocular irritation, resulting in a mean in vitro irritancy score of -0,29 after 10 minutes of treatment. The calculated In Vitro Irritancy Score was less than 55, indicating that the test article did not cause serious eye damage, and thereby ruling out GHS Category 1.

In order to reach a definitive result for acute eye hazard classification , the EpiOcular™ Eye Irritation Test was performed

EIT

MatTek EpiOcular™ tissue samples were treated in duplicate with the test article, negative control and positive control for 30 minutes. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 31.2% after 30+-2 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of two tissues treated with the test item was less than 8%, indicating that the test system functioned properly.

Eye hazard potential is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 30+-2 minutes treatment with the test item compared to the negative control tissues was 94.3%. Since the mean relative tissue viability for the test item was greater than 60% after 30 +- 2 minutes treatment it is considered to be non-irritant to the eye.

In conclusion the test item is identified as not requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Justification for classification or non-classification

The results from these key studies support the conclusions that no classification is warranted for skin and eye irritation/corrosion according to CLP Regulation 1272/2008.