Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 June 2018 - 8 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 431

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

EC Number:

268-417-2

EC Name:

3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Cas Number:

68084-04-8

Molecular formula:

C13H19N

IUPAC Name:

3-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbonitrile

Constituent 2

Reference substance name:

4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

EC Number:

244-530-2

EC Name:

4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Cas Number:

21690-43-7

Molecular formula:

C13H19N

IUPAC Name:

4-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbonitrile

Test material form:

liquid

Specific details on test material used for the study:

Test Material: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile
Public name: Azuril
EC number: 915-371-2 (pre-registration 244-530-2)
CAS number: n/a (pre-registration 21690-43-7)
Batch/Lot number: A170421E
Appearance: Clear, pale yellow liquid
Purity: 99.35 %
Expiry date: 06 June 2019

Storage conditions: Room temperature (15-25 °C, ≤ 70 % relative humidity (RH)), under inert gas, protected from humidity (tightly closed container)
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.
Indentification and Receipt: The test item of a suitable chemical purity was supplied by the Sponsor.
Preparation of the Test Item: The test item was applied as supplied.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.:EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-023, Expiry Date: 11 June 2018)

Source strain:

other: NA

Details on animal used as source of test system:

TEST SYSTEM: EPISKINTM(SM)
EPISKINTM(SM) is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Ref: (Tinois et al., 1994).

Justification for test system used:

The EPISKINTM(SM) model has been validated for corrosivity and irritation testing in an international validation study. This is recommended by the relevant OECD guidelines for corrosivity and irritation testing (OECD No. 431 and OECD No. 439); for this reason, it was considered to be suitable for this study.

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EPISKIN TM (SM) (Manufacturer: SkinEthic, France, Batch No.: 18-EKIN-023, Expiry Date: 11 June 2018)
- Units: EPISKIN TM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm*2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plates
- Punch: EPISKIN TM (SM) biopsy punch for easy sampling of epidermis
- A flask of sterile “Assay Medium” (Batch No.: 18 ESSC 024; Exp. Date: 13 June 2018)
- Storage: The kits were kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8 °C until the initiation of the test.

MTT SOLUTION
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solutions were stored in refrigerator (2-8 °C) protected from light. These were diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

ACIDIFIED ISOPROPANOL
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.

CHEMICALS USED IN THE EXPERIMENT:
- MTT
- Isopropanol
- Hydrochloric acid
- Phosphate buffered saline (PBS)

PREDICTION MODEL / DECISION CRITERIA
General validity criteria
- The mean OD value of the two or three negative control tissues should be ≥ 0.6 and ≤ 1.5.
- The mean OD value of the blank samples (acidified isopropanol) should be < 0.1.
Specific criteria for corrosivity
- The difference of viability between the two tissue replicates should not exceed 30%.
- The acceptable mean percentage viability range for positive controls is ≤ 20%.

Amount/concentration applied:

TEST MATERIAL
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath.

50 µL of test item was applied evenly to each of two test units and each additional control skin units.

NEGATIVE AND POSITIVE CONTROLS
50 µL of negative control (Physiological saline) or positive control (glacial acetic acid) were added to each skin unit

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 4 hours at room temperature (21.9-23.4°C).

Number of replicates:

TEST ITEM TEST AND CONTROLS:
- Two replicates per time point were used for test item
- Two negative controls and two positive controls
- Two additional test item-treated living tissues were used for the non-specific OD evaluation

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

122.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of skin corrosion

Remarks:

Mean Viability Exp 1,2 (%): 128.8

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

134.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no indication of skin corrosion

Remarks:

Mean Viability Exp 1,2 (%): 128.8

Other effects / acceptance of results:

VALIDITY OF THE TEST
General validity criteria:
- After receipt, the two indicators of the delivered kits were in proper conditions.
- The mean OD value of the two negative control tissues in the corrosivity and irritation test was in the recommended range (0.954 and 0.723).
- The mean OD value of the blank samples (acidified isopropanol) was 0.047 in both cases.

Specific criteria for corrosivity testing:
- The two positive control treated tissues showed 0.3% viability demonstrating the proper performance of the assay.
- The difference of viability between the two test item-treated tissue samples in the MTT assay was 9.1%.
- The difference of viability between the two negative control tissue samples in the MTT assay was 0.5%.


All these parameters were within acceptable limits and therefore the study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results of this study indicated that test material Azuril [Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile] is non-corrosive to the skin.

Executive summary:

The aim of this study was to evaluate the corrosion potential to skin of the test material Azuril (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) using reconstructed the human epidermis model EPISKINTM(SM). The study was performed according to the OECD 431 Guideline and under GLP conditions.

 

Disks of EPISKINTM(SM) were treated with the test item and incubated 4 hours at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a > 95% humidified atmosphere. Physiological saline (0.9% (w/v) NaCl solution) treated epidermis were used as negative control and glacial acetic acid treated epidermis were used as positive control (two units/control)

 

The result of this study showed that, following exposure with Azuril[Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile], the mean cell viability was 128.8% relative viability compared to the negative control. This value is above the threshold of 35%, therefore the test item was considered non-corrosive to skin. The validity criteria were met and the study was considered to be valid.