Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

Endpoint summary

Description of key information

In conclusion, it can be stated that in this study and under the reported experimental conditions, H-32 is non corrosive to skin according to EU CLP and UN GHS.

In conclusion, it can be stated that in this study and under the experimental conditions reported, H-32 is not irritant to skin according to UN GHS and EU CLP regulation.

Relative to the negative control, the test item H-32 did not cause a relevant increase of the corneal permeability. The calculated mean in vitro irritancy score was 2.11. According to OECD 437 the test item is not categorized (GHS).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 February 2017-08 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Not specified

Source strain:

not specified

Details on animal used as source of test system:

Epi-200 kits and MTT-100 assays were purchased from MatTek Corporation (Bratislava, Slovakia). The EpiDerm™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ∅).
EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate and reached Envigo CRS GmbH on 07 March 2017. On day of receipt the preincubation phase of the EpiDerm™ tissues started.

Vehicle:

unchanged (no vehicle)

Details on test system:

At least 1 hour before dosing, EpiDerm™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. A 24-well plate was prepared as holding plate containing 300 μL assay medium. The holding plate was pre warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) until use.

Duplicate EpiDermTM tissues were treated with the test item, positive control or negative control for the following exposure times:
• Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
• Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
• Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes
After the pre-incubation of the EpiDermTM tissues was completed the DMEM-based medium in each well was replaced with 0.9 mL fresh assay medium. The 6-well plates for the 3 ± 0.5 minutes exposure periods stayed at room temperature in the sterile bench, the 6-well plates for the 60 ± 5 minutes exposure period were placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle / multipipette containing DPBS to remove any residual test material (20 times). Excess DPBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

Two 24-well plates were prepared prior to the end of the tissue pre-warming period. MTT solution (300 μL) was added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until required.
Following rinsing, the tissues were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the tissues were rinsed three times with DPBS and carefully dried with blotting paper. The inserts were transferred into new 24-well plates. The tissues were each immersed in 2 mL of extractant solution (isopropanol) pipetted in each well ensuring that the tissues were completely covered. The 24-well plates were sealed to minimise isopropanol evaporation. The formazan salt was extracted for 2 hours while shaking at room temperature. After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24- well plates were then placed on a shaker for 15 minutes until the solution was homogeneous in colour. 3 × 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96- well flat bottom microtiter plate. The OD was determined in a microplate reader (Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL (79.4 μL/cm2 according to guideline) of the test item were dispensed directly onto duplicate EpiDermTM tissue surface.
Concurrent controls were used for several Envigo CRS GmbH studies performed simultaneously. Each 50 μL were applied to each set of duplicate tissues for the 3 min and 1 hour exposure periods.

Duration of treatment / exposure:

3 minutes and 60 minutes

Irritation / corrosion parameter:

other: relative absorbance value %

Run / experiment:

3 mins exposure

Value:

95.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

other: relative absorbance value %

Run / experiment:

60 mins exposure

Value:

90.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

This in vitro study was performed to assess the corrosive potential of H-32 by means of the Human Skin Model Test with EpiDerm™ tissues models. The test item passed the MTT- and the Colour Interference pre-tests. Independent duplicate tissues of EpiDermTM were exposed to the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour,

respectively. Afterwards, the test and the control items were rinsed off the tissues, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. MTT solution was then

aspirated from the wells and the wells were rinsed with DPBS. Inserts were transferred into new 24 well plates. The formazan salt was extracted for 2 hours at room temperature.

The required acceptability criteria were met. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period (25.8%) and for the 1 hour exposure period (2.3%) thus confirming the validity of the test system and the specific batch of tissue models. After exposure to the test item H-32 the relative absorbance value only decreased slightly to 95.9% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 90.7%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, H-32 is non corrosive to skin according to EU CLP and UN GHS.

Executive summary:

This in vitro study was performed to assess the corrosive potential of H-32 by means of the Human Skin Model Test with EpiDerm™ tissues models.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye deionised water or changed colour when mixed with it (pre-test for colour interference).

Consequently, additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary. Independent duplicate tissues of EpiDerm™ were exposed to either the test item, the negative control (deionised water) or the positive control (8.0 N KOH) for 3 minutes and 1 hour, respectively.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the

acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The CV in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus

the validity of the test system and the specific batch of the tissue models is confirmed. After exposure of the tissues to the test item the relative absorbance value decreased to 95.9% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 90.7%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive. In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item H-32 is non corrosive to skin according to EU CLP and UN GHS.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 March 2017-17 March 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

N/A

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:Human Skin Model Test
- Tissue batch number(s):17-EKIN-011
- Date of initiation of testing: 14 March 2017
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 14 March 2017.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues
were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for about 3.3 hours at room temperature with gentle agitation.


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
Prior to the start of the test, the test item’s colour interference potential had to be evaluated. For this purpose the test item (10 μL) was mixed with 90 μL of deionised water in a preexperiment. The test item/water mixture was gently shaken for 15 minutes at room temperature. The water was not dyed by the test item and the colour of the test item/water mixture did not change during the incubation period compared with the colour of the pure test item. Therefore, the measurement of the OD of the test item in water at 570 nm was not required and consequently not performed. An additional test with viable tissues (without MTT
addition) to determine a correction factor for calculating the true viability in the main experiment did not have to be performed. For correct interpretation of results it is necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 μL of the test item was added to 2 mL of
MTT solution (0.3 mg/mL) and the mixture was incubated in the dark at 37 ± 1.5 °C (5 ± 0.5% CO2) for 3 hours. MTT solution was used as the control. If the MTT
solution colour turns blue/purple, the test item is presumed to have reduced the MTT. Since the colour did not turn blue/purple, the test item was not considered to be a MTT reducer. An additional test with freeze-killed tissues to determine a correction factor for calculating the true viability in the main experiment did not have to be performed.


NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : 3
- Method of calculation used:

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

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):10 μL (26.3 μL/cm2) of the undiluted test item were applied to each of the triplicate tissues.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):Each10 μL were applied to each of triplicate tissues for 15 minutes.
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight):Each10 μL were applied to each of triplicate tissues for 15 minutes.

Duration of treatment / exposure:

15 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Average of 3 runs

Value:

87.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

This in vitro study was performed to assess the irritation potential of H-32 by means of the Human Skin Model Test.

The test item passed the pre-tests for direct MTT reduction and colour interference. Each three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. The test item and the positive and negative controls were washed off the skin tissues treatment. After further incubation for about 42.6 hours the tissues were treated with the MTT

solution for 3 hours following approximately 3.3 hours extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm. After treatment with the negative control the absorbance values were well within the required

acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 (values between 0.786 and 0.930) for the 15 minutes treatment interval thus showing the quality of the tissues. The data of the quality control (determined by SkinEthic Laboratories, 69007 Lyon, France) of the respective EpiSkin™ lot is mentioned in the present report (see annex 2; the acceptance limit of the IC50 should be between 1.5 and 3.0 mg/mL after 18 hours treatment with SLS; true value: 2.0 mg/mL).

Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.9% thus ensuring the validity of the test system. The rel. standard deviations between tissues of the same treatment group was below 9%

(threshold of the "OECD TG 439 Guideline for the Testing of Chemicals: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

After treatment with the test item the mean relative absorbance value was reduced to 87.6%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, H-32 is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

This in vitro study was performed to assess the irritation potential of H-32 by means of the Human Skin Model Test according to OECD TG 439.

The test item did not reduce MTT (pre-test for direct MTT reduction), and it did not dye water or did not change colour when mixed with it (pre-test for colour interference). Consequently, additional tests with freeze-killed or viable (without MTT addition) tissues to determine correction factors for calculating the true viability in the main experiment were not

necessary. Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative control (deionized water) or the positive control (5% sodium lauryl sulfate) for 15 minutes. After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 thus showing the quality of the tissues. Treatment with the positive control induced a sufficient decrease in the relative absorbance as

compared to the negative control thus ensuring the validity of the test system. After treatment with the test item the mean relative absorbance value increased to 87.4%. This value is above the threshold for irritancy of > 50%. Therefore, the test item is not considered to possess an irritant potential. In conclusion, it can be stated that in this study and under the experimental conditions reported, the H-32 is not irritant to skin according to UN GHS and EU CLP regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2017-20 February 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in HBSS. The corneae were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

The anterior compartment received the test item or negative or positive control at a volume of 0.75 mL on the surface of the corneae.

Duration of treatment / exposure:

The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted ten minutes.

Duration of post- treatment incubation (in vitro):

After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130).

Number of animals or in vitro replicates:

9 replicates in total.
3 for the negative control
3 for the positive control
3 for the test item.

Details on study design:

All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0). The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and the negative and positive controls.

The anterior compartment received the test item or negative or positive control at a volume of 0.75 mL on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted ten minutes. After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment. Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130). In the second step of the assay, permeability of the corneae was determined.

The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea. After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130).

Following the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer. The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490).

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean value

Value:

2.11

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

This in vitro study was performed to assess the corneal damage potential of H-32 by means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the neat test item H-32, the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in incubation medium, and opacity was measured a second time (t130). After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.92). The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 79.74) corresponding to a classification

as serious eye damaging (CLP/EPA/GHS (Cat 1)). The test item H-32 was tested undiluted. Relative to the negative control, the test item H-32 did not cause a relevant increase of the corneal permeability. The calculated mean IVIS was 2.11 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437, the test item is not categorized.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, H-32 is not categorized (GHS).

Executive summary:

This in vitro study was performed to assess the corneal damage potential of H-32 by means of the BCOP assay using fresh bovine corneae. After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation

medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards, opacity was measured a second time (t130). After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). Relative to the negative control, the test item H-32 did not cause a relevant increase of the corneal permeability. The calculated mean in vitro irritancy score was 2.11. According to OECD 437 (see table in chapter 3.8.3) the test item is not categorized (GHS).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

All studies indicate that the substance does not meet criterai for classification as a skin irritant, corrosive to skin or irritating to eyes.

The substance is classified as not hazardous to skin and eyes.