dipropyl cyclohexane-1,2-dicarboxylate

Endpoint summary

Description of key information

The test substance, CHP, elicited a mean tissue viability of 107.6 ± 3.6% and was predicted as non-irritant to the skin, classification, No category.

The test substance, CHP, elicited an In Vitro Irritancy Score of 0.3 ± 0.6 and was predicted to have a classification of No Category according to the UN GHS.

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:

29th July 2015 -28th August 2015

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Reconstructed Human Epidermis Test Method, Guideline 439, 26 July 2013.

Qualifier:

according to guideline

Guideline:

other: In Vitro Skin Irritation Test: Human Epidermis Model EpiSkin™, EpiSkin™ Skin Irritation Test15 min - 42 hours – February 2009 Version 1.8, supplied by L’Oreal (leading laboratory in the validation of the test for ECVAM).

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

The EpiSkin™ Skin Irritation Test15 min - 42 hours using reconstructed human epidermis skin constructs supplied by SkinEthic Laboratories, Lyon, France, was accepted as a replacement to the in vivo Draize Skin Irritation Test, by the European Centre for the Validation of Alternative Methods (ECVAM) in a statement dated 27 April 2007.

The test involves the application of the test substance for 15 minutes to the EpiSkin™ threedimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1
matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38cm2. The EpiSkin™ kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.

The principle of the assay is that irritant substances are sufficiently cytotoxic to cause cell damage in the cell layers. The cell viability is determined bymitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify
irritant and non-irritant substances. The test includes acceptance criteria for both negative and positive controls.

Control samples:

other: The negative control was sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium. The positive control was 5% Sodium Dodecyl Sulphate (SDS) in purified water.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):The test substance, CHP, a colourless liquid, positive and negative controls were in liquid
form and were applied by dispensing a volume of 10 micro litres over each tissue using a positive displacement pipette.

Duration of treatment / exposure:

After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 +/- 0.5 minutes with the test substance, negative orpositive control at room temperature. A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 7 minutes application time.

After 15 +/- 0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate
Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 +/- 1 hour at 37 +/- 2°C in a humidified atmosphere of 5% CO2 in air.

After 42+/- 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours+/- 5 minutes at 37 +/- 2°C in a humidified atmosphere of 5% CO2 in air.

At the end of 3 hours +/- 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro-tube.

When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration).

The tissues were extracted over 4 hours, protected from light at room temperature (vortexing after approximately 2 hours).
After formazan extraction, duplicate 200 μL aliquots of the extractant from each tube were pipetted into the wells of flat-bottomed 96-well plates. Theextractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

Irritation / corrosion parameter:

other: other: Mean tissue viability

Value:

107.6

Remarks on result:

other:

Remarks:

Basis: other: Episkin. Reversibility: no data. (migrated information)

Assessment of viability

The mean Optical Density (OD) for the six replicate blanks was subtracted from the individual substance and control tissues OD.

The viability of each tissue was expressed as a percentage of the mean negative control value.

Assay acceptance criteria

Negative control

The OD from the negative control tissue in the MTT assay is an indicator of tissue viability after the shipping and storage procedure and under the specific conditions of the assay. The mean absorbance of the triplicate negative control values should be >=0.6 and ≤1.5. The Standard Deviation (SD) value of the % viability should be ≤18.

Positive control

The OD of the positive control is an indicator of the sensitivity of the tissues. The mean viability should be ≤40% of the negative control and the SD ≤18%.

Data interpretation - Prediction model

If the mean tissue viability was equal to or less than 50% of the negative control value, the sample was classed as Category 2 (GHS classification).

Criteria for in vitro interpretation	Globally Harmonized System
Mean tissue viability is ≤ 50 %	Category 2
Mean tissue viability is > 50 %	No category

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance, CHP, elicited a mean tissue viability of 107.6 ± 3.6% and was predicted as non-irritant to the skin, classification, No category.

Executive summary:

The objective of this test was to assess the skin irritation potential, in vitro, of the test substance, CHP.

The test substance was applied to EpiSkin™ human epidermis skin constructs. The constructs consisted of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum. The constructs were treated with the neat test substance for 15 minutes. After rinsing of the test substance the constructs were incubated for 42 hours. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.

This assay was valid with negative and positive controls showing results within the acceptable range.

The test substance, CHP, elicited a mean tissue viability of 107.6 ± 3.6% and was predicted as non-irritant to the skin, classification, No category.

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:

4th August 2015 - 5th August 2015

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The bovine eyes, supplied by Joseph Morris Abattoir, were excised by an abattoir employee and collected as soon after slaughter as possible (last excised at 13:15 hours, 4 August 2015).

Vehicle:

physiological saline

Controls:

not specified

Amount / concentration applied:

The test substance, CHP, was diluted to 10% with 0.9% saline.

Duration of treatment / exposure:

The test substance and the positive control were tested undiluted.

Immediately prior to treatment, the medium was removed from the anterior compartment of the holder using a pipette tip attached to a vacuum pump, taking extra care to ensure all excess liquid had been removed. Seven hundred and fifty μL (750 μL) of the test substance, positive control or negative control was introduced into the anterior part of each holder. Following application, the anterior compartment was plugged and the holder turned to a horizontal position and slightly rotated to ensure uniform distribution of the test substance over the surface of the cornea. The test substance or controls were in contact with the cornea for a total of 10 minutes (+/-30 seconds). Each holder was incubated in a horizontal position at 32°C +/- 1°C in a waterbath.

Duration of post- treatment incubation (in vitro):

two hours +/- 10 minutes at 32°C +/- 1°C.

Number of animals or in vitro replicates:

Corneas were treated in triplicate with either the test substance, positive control (ethanol) or negative control (0.9% sodium chloride solution).

Details on study design:

Following incubation, the test substance, positive and negative controls were removed from the epithelial surface of the cornea by washing, at least three times or until the wash medium (EMEM with phenol red) was clear and there was no discolouration. The wash medium was added via the holes on the top of the holder. After each wash, the wash medium was removed using a pipette tip attached to a vacuum pump. The test substance required four washes. The anterior compartment was then filled with cMEM taking care to ensure no air bubbles were present in the compartment. Once all the air bubbles had been removed, the anterior compartment was re-plugged and the holders returned to the waterbath and incubated, in an upright position, for two hours +/- 10 minutes at 32°C +/- 1°C.

Following completion of the two hour incubation period, the medium was removed from both compartments and replaced with fresh cMEM. The posterior compartment was re-plugged and the opacity of each cornea measured and recorded. The opacity values obtained at this stage were used in calculating the final In Vitro Irritancy Score.

Throughout the assay the corneas were examined for opaque spots or other irregularities. The sodium fluorescein stock solution (4 mg/mL) was thawed prior to commencing the permeability incubation. Following the final opacity measurement, the medium was removed from the anterior compartment of the holder. One mL (1 mL) of the sodium fluorescein solution was added to the anterior compartment using a micropipette.
Following addition of the sodium fluorescein solution to the anterior side of the holder, the compartment was plugged and the corneas incubated in a horizontal position at 32°C ± 1°C for 90 ± 5 minutes in a waterbath. Following incubation, the medium in the posterior compartment was mixed by drawing approximately 2.5 mL gently up and down a 5 mL syringe, with a needle attached, three times. An aliquot of the mixed medium from the posterior compartment was removed and transferred to a 1 cm path length cuvette. A spectrophotometer was adjusted to read at 490 nm (OD490) and a sample of cMEM read (OD = 0.062).


The positive control should elicit an In Vitro Irritancy Score that falls within two standard deviations of the historical mean for the laboratory. The negative control mean opacity change value should be ≤3.0 and the permeability mean value ≤0.1.

Irritation parameter:

in vitro irritation score

Value:

0.3

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance, CHP, elicited an In Vitro Irritancy Score of 0.3 ± 0.6 and was predicted to have a classification of No Category according to the UN GHS.

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the

potential of the test substance, CHP, to cause serious eye damage. Ethanol was tested in

parallel as a positive control.

The BCOP assay is an organotypic model that uses isolated bovine corneas from freshly

slaughtered cattle as a means of assessing the potential of a test substance to cause serious

eye damage. Two endpoints, corneal opacity and permeability, were measured and combined

to give an In Vitro Irritancy Score which was used to assign an in vitro irritancy hazard

classification category for prediction of the ocular irritation potential of the test substance.

The negative and positive controls met the acceptance criteria for this assay.

The test substance, CHP, elicited an In Vitro Irritancy Score of 0.3 ± 0.6 and was predicted to

have a classification of No Category according to the UN GHS

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

The objective of this test was to assess the skin irritation potential, in vitro, of the test substance, CHP.

The test substance was applied to EpiSkin™ human epidermis skin constructs. The constructs consisted of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum. The constructs were treated with the neat test substance for 15 minutes. After rinsing of the test substance the constructs were incubated for 42 hours. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.

This assay was valid with negative and positive controls showing results within the acceptable range.

The test substance, CHP, elicited a mean tissue viability of 107.6 ± 3.6% and was predicted as non-irritant to the skin, classification, No category.

Justification for classification or non-classification

The test substance, CHP, elicited a mean tissue viability of 107.6 ± 3.6% and was predicted as non-irritant to the skin, classification, No category.

The test substance, CHP, elicited an In Vitro Irritancy Score of 0.3 ± 0.6 and was predicted to have a classification of No Category according to the UN GHS.