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

Endpoint summary

Description of key information

Skin corrosion (OECD 431): non-corrosive

Skin irritation (OECD 439): non-irritant

Eye irritation (OECD 438): non-irritant

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:

6 June 2018 - 8 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 431

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)

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.

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

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

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.

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.

Reference 2

Endpoint:

skin irritation: 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 439

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

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 “Maintenance 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.

10 µL of test item were applied evenly to each of three test units and each additional control skin units. The test item was spread gently on the skin surface with a pipette tip without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

NEGATIVE AND POSITIVE CONTROLS
50 µL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette in the irritation test

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.1-23.6°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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

85.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Mean Viability Exp 1,2,3 (%): 90.8

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

78.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Mean Viability Exp 1,2,3 (%): 90.8

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

108.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Viability Exp 1,2,3 (%): 90.8

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 irritation test was in the recommended range (0.676).
- The mean OD value of the blank samples (acidified isopropanol) was 0.048 in both cases.

Specific criteria for irritation testing:
- Standard deviation of the viability results for negative control samples was 0.2%.
- The positive control treated tissues showed 8.9% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 2.7%.
- The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 15.9%.

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

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-irritant to the skin.

Executive summary:

The aim of this study was to evaluate the potential 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) to be irritant to skin. The experiment was conducted using the reconstructed the human epidermis model EPISKINTM(SM). It was performed according to the OECD 439 Guideline and under GLP conditions.

 

The EPISKINTM(SM) disks were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a > 95% humidified atmosphere. 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. PBS treated epidermis were used as negative control and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive 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 90.8% relative viability compared to the negative control.

 

This value is above the threshold of 50%, therefore the test item was considered non-irritant to skin. The validity criteria were met and the study was considered to be valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN CORROSION

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.

SKIN IRRITATION

The aim of this study was to evaluate the potential 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) to be irritant to skin. The experiment was conducted using the reconstructed the human epidermis model EPISKINTM(SM). It was performed according to the OECD 439 Guideline and under GLP conditions. The EPISKINTM(SM) disks were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a > 95% humidified atmosphere. 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. PBS treated epidermis were used as negative control and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as positive 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 90.8% relative viability compared to the negative control. This value is above the threshold of 50%, therefore the test item was considered non-irritant to skin. The validity criteria were met and the study was considered to be valid.

 

EYE IRRITATION

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (09 October 2017). After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with 30 μL of the undiluted test item. The three positive control eyes were treated in a similar way with 30 μL of 5% (w/v) Benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated. The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in the experiment. Thus, the experiment was considered to be valid. No significant corneal swelling (0 to -5%) was observed during the four-hour observation period on the test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on three eyes. Slight fluorescein retention change (severity 0.5 on two eyes and severity 1 on one eye) was noted in all three eyes. No other corneal effect was observed. Based on this in vitro eye irritation in the isolated chicken eyes test 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 test item is non-irritant, UN GHS Classification: No Category.

Justification for classification or non-classification

Based on the results of the eye and skin irritation/corrosion studies, Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile (Azuril) is non-irritant/corrosive to skin and non-irritant to eyes, and does not meet the criteria to be classified under EU Regulation (EC) No 1272/2008 (CLP).