Methyl 2-hexyl-3-oxocyclopentanecarboxylate

Endpoint summary

Description of key information

The test substance is not irritating to the skin.

The test substance is not irritating to the eye.

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

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)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Description: Colourless to pale yellow liquid
Storage Conditions: Room temperature, protected from light
Expiry Date: 18 January 2020

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

- Preparation and Application of the Test Item: Prior to treatment, all EpiDerm tissues were gently blotted to remove moisture. The test item was applied undiluted. 30 μL (47 μL/cm2) of the test item were dispensed directly atop the EpiDerm tissue. The test item was gently spread to match size of the tissue using a bulb-headed Pasteur pipette.
- Controls: Controls were set up in parallel to the test item cultures in order to confirm the validity of the test. Negative Control: Dulbecco’s phosphate buffered saline; Positive Control: TC-SDS-5%
- Dose Groups: 1. Negative control 30 μL DPBS; 2. Positive control 30 μL 5% SDS solution; 3. Test Item 30 μL (undiluted). The test was performed on a total of 3 tissues per dose group.
- Test System: The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.
- Pre-Experiments: To check the non-specific MTT-reducing capability of the test item 30 μL of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator. To check the colouring potential of the test item 30 μL of the test item were mixed per 300 μL aqua dest. and per 300 μL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min.
- Experimental Procedure: Upon receipt of the EpiDerm, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL assay medium per well. The surface was dried using a sterile cotton tip and the plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 for 60 ± 5 min. Subsequently the tissues were transferred into new wells containing 0.9 mL pre-warmed assay medium per well and were incubated for 18 ± 3 h in a humidified incubator at 37 ± 1 °C, 5.0% CO2. After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, e.g. in one-minute intervals. After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow for the remaining time until the 60 ± 1 min incubation time of the first dosed tissue was over. Then the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper. The inserts were placed in prepared new 6-well plates containing 0.9 mL pre-warmed fresh assay medium per well and the tissue surface was dried using a sterile cotton tip. The plates were post-incubated at 37 ± 1 C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h. After this post-incubation period the bottom of the inserts were blotted on sterile blotting paper and the inserts were transferred in a prepared 24-well plate containing 300 μL pre-warmed MTT medium. This plate was incubated for 3 h ± 5 min at 37 ± 1 °C, 5.0% CO2, humidified to 95%. After the MTT incubation period, the tissues were rinsed three times with DPBS and afterwards placed on blotting paper to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 h with gentle shaking on a plate shaker. Before using the extracts, the plate had been shaken for at least 15 min on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 μL aliquots per each tissue were transferred into a 96-well plate. OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.
- Data Analysis: Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”), if the tissue viability after exposure and post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.
- Test Acceptance Criteria: The test meets acceptance criteria if: mean absolute OD570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8; mean relative tissue viability of the three positive control tissues is ≤ 20%; standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

Irritation / corrosion parameter:

% tissue viability

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Pre-Experiments: The mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixture of 30 μL of the test item per 300 μl aqua dest. and/or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.
Controls: The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.385). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (2.5%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (1.4% - 15.4%).

Interpretation of results:

GHS criteria not met

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

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)

Version / remarks:

Adopted 9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Description: Colourless to pale yellow liquid
Storage conditions: Room temperature, protected from light
Expiry date: 18 January 2020

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Abattoir A. Moksel AG, Buchloe, Germany.
- Storage, temperature and transport conditions of ocular tissue: On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
- Time interval prior to initiating testing: The corneas were incubated for one hour at 32 ± 1 °C
- indication of any existing defects or lesions in ocular tissue samples: Any defective cornea had been discarded.
- Indication of any antibiotics used: Pen/Strep

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 μL of the test substance or the control substance was introduced into the anterior chamber.

Duration of treatment / exposure:

10 minutes incubation at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

- Illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C.
- Optical density at 490 nm (OD490) was determined, 90 minutes after the illuminance measurement was performed.

Number of animals or in vitro replicates:

Triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay.

NUMBER OF REPLICATES
Triplicates

NEGATIVE CONTROL USED
As negative controls physiological saline (0.9% NaCl) was used.

POSITIVE CONTROL USED
As positive controls ethanol (100%) was used.

APPLICATION DOSE AND EXPOSURE TIME
The medium was removed from the anterior chamber and replaced with the test item or control. 750 μL of the test substance or the control substance was introduced into the anterior chamber. Exposure time was 10 minutes incubation at 32 ± 1 °C.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).
- POST-EXPOSURE INCUBATION: The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Optical density at 490 nm (OD490) was determined, 90 minutes after the illuminance measurement was performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: An initial measurement was performed on each of the corneas using the opacitometer. The illuminance of each cornea was read and recorded before and after treatment.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: See explanation and Table 1 in ‘Any other information on materials and methods incl. tables’.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean based on #1, #2, #3

Value:

0.06

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Run / experiment:

Mean #1, #2, #3

Value:

0.15

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein leakage

Run / experiment:

Mean #1, #2, #3

Value:

-0.006

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL EPITHELIUM CONDITION
None of the 3 corneas treated with Dihydro Isojasmonate showed opacity of the tissue.

CRITERIA FOR AN ACCEPTABLE TEST
The positive control In Vitro Irritancy Score was 42.99. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid (see Table 4 in 'Any other information on results incl. tables'.)
The negative control gave opacity of ≤0.77 and permeability ≤0.027. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Individual and mean corneal opacity, permeability measurements and invitro irritation scores can be found in Table 1-3 in 'Any other information on results incl. tables'.

Table 1. Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1		1.11	1.61	0.50
2	Negative	1.11	1.87	0.75
3	Control	1.11	2.16	1.05
MV		1.11	1.88	0.77
4		3.19	34.99	31.80	31.03
5	Positive	2.16	29.80	27.64	26.87
6	Control	2.27	29.49	27.22	26.45
MV		2.54	31.43	28.89	28.12
7		-0.51	1.04	1.55	0.79
8	Test Item	-0.61	-0.21	0.39	-0.38
9		-0.15	0.66	0.81	0.04
MV		-0.42	0.50	0.92	0.15

MV = mean value

 

Table 2. Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1		0.022
2	Negative	0.045
3	Control	0.015
MV		0.027
4		0.985	0.958
5	Positive	1.166	1.139
6	Control	0.905	0.878
MV		1.019	0.991
7		0.020	-0.007
8	Test Item	0.019	-0.008
9		0.025	-0.002
MV		0.021	-0.006

MV = mean value

 

Table 3. In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1		0.50	0.022
2	Negative	0.75	0.045
3	Control	1.05	0.015
MV		0.77	0.027	1.18
4		31.03	0.958
5	Positive	26.87	1.139
6	Control	26.45	0.878
MV		28.12	0.991	42.99
7		0.79	-0.007
8	Test Item	-0.38	-0.008
9		0.04	-0.002
MV		0.15	-0.006	0.06

MV = mean value

 

Table 4. Historical Mean In Vitro Irritation Score of the Positive Control

	IVIS Positive Control - Ethanol 100 %
Mean Value (MV) Standard Deviation (SD) MV- 2xSD MV+2xSD	48.57 9.69
	29.20 67.94
Number of Replicates providing Historical Mean: 50

Positive controls are updated after every single experiment or at least every 3 months

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin irritation

Skin irritation was investigated in an in vitro skin irritation test using the Reconstructed Human EpiDerm™-Standard Model (EPI-200) according to OECD Guideline 439 and following GLP. The test item was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%. The mixture of 30 μL of the test item per 300 μL aqua dest. and/or per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%. The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.385). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (2.5%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (1.4% - 15.4%). The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (100.0%) after 60 min treatment and 42 h post-incubation.

  

In vitro eye irritation

The eye irritancy potential of the test item was investigated in the bovine corneal opacity and permeability assay according to OECD 437 and following GLP. The assay uses isolated bovine corneas obtained as a by-product from animals freshly slaughtered. The test was performed in triplicates. The medium was removed from the anterior chamber and replaced with the test item or control. The corneas were exposed to 750 μL the test substance or the control substance (physiological saline as negative control and 100% ethanol as positive control). After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). Illuminance measurement was performed after 2 hours of incubation at 32 ± 1 °C. Also, each cornea was observed visually and pertinent observations were recorded. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were icubated for 90 minutes at 32 ± 1 °C. Then the illuminance measurement was performed, optical density was assessed using a spectrophotometer (OD490).

None of the 3 corneas treated with the test ietm showed opacity of the tissue. The mean in vitro irritating score for the test substance was 0.06. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

In conclusion, this Bovine Corneal Opacity and Permeability test (BCOP) indicated that the test item is not considered to be irritating to the eye.

Justification for classification or non-classification

Based on the available information, classification of the test substance for skin or eye irritation is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.