5-methyl-2-(isopropyl)cyclohexyl nicotinate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Principles of method if other than guideline:

The toxic occular irritation potential of the substance was evaluated in the cytotoxicity assay (NRU - Neutral Red Uptake) by using Balb 3T3 clone 31 cells.

GLP compliance:

no

Remarks:

existing studies not conducted in GLP but well documented

Test material

Test material information

Constituent 1

Reference substance name:

5-methyl-2-(isopropyl)cyclohexyl nicotinate

EC Number:

254-991-1

EC Name:

5-methyl-2-(isopropyl)cyclohexyl nicotinate

Cas Number:

40594-65-8

Molecular formula:

C16H23NO2

IUPAC Name:

5-methyl-2-(isopropyl)cyclohexyl nicotinate

Test system

Vehicle:

other: emulsified in corn oil and diluted in culture medium.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 2000; 1000; 500; 250; 125; 62.5; 31.3; 15.6 μg/ml.

VEHICLE: the test material was first emulsified in corn oil and then diluted in culture medium.

Duration of treatment / exposure:

24 hours

Details on study design:

- Cell model used: fibroblasts Balb 3T3 clone 31cells.
- Number of repetitions and replicates used
- Test chemical concentrations used: 2000; 1000; 500; 250; 125; 62.5; 31.3; 15.6 μg/ml.
- Duration of exposure to the test chemical: 24 hours.
- Exposure conditions: 37 °C and 5 % CO2.
- Description of the procedure: the substance was added in the wells containing the cells. After the incubation period for 24 hrs, medium containing tested product was removed. The cells then washed with PBS and fresh culture medium, containing Neutral Red dye; was added to the wells. Cells were incubated at 37 °C and 5 % CO2 for 3 hours. At the end of this period NR-medium was removed, cells were washed with PBS and a solution to solubilize NR captured by cells. When solubilisation was completed, well plate was read at 540 nm.
Untreated cells are used as negative control. Cells treated with SDS scalar concentrations from 250 ug/ml to 15.6 ug/ml are used as positive control.
- Calculation of cell viability: for each tested product concentration, % cell viability was calculated according to the formula: % CELL VIABILITY = (OD540 treated *100)/OD540 negative control. Recorded mortality data were plotted against respective product concentrations to create a dose- response curve. By using curve equation IC50 parameter (or IC50, Inhibiting concentration 50), the product concentration that causes mortality in 50 % of cell population, was calculated.
- Description of evaluation and decision criteria used: If the NR50 > 1000 μg/ml the Modified Maximum Average Score (MMAS) is < 25 and the substance is considered as no/mild irritant. If the NR is between 15 μg/ml and 1000 μg/ml, the MMAS is 80.46-logNR50 and is considered as a moderate irritant. If the NR50<15 μg/ml then the MMAS is >60 and the substance is considered as a strong irritant.

Results and discussion

In vitro

Results

Irritation parameter:

other: NR50 (μg/ml)

Value:

> 1 000

Vehicle controls validity:

valid

Remarks:

NR50 > 1000 μg/ml

Positive controls validity:

valid

Remarks:

NR50 = 0.099 μg/ml

Remarks on result:

no indication of irritation

Any other information on results incl. tables

The cell viability of the sample is presented in the table below for each concentration.

Table

Concentration μg/ml	Cell viability %	% cell mortality
2000	90.57	9.43
1000	91.80	8.20
500	94.19	5.81
250	96.14	3.86
125	96.35	3.65
62.5	96.93	3.07
31.3	103.50	-3.50
15.6	103.79	-3.79

According to the obtained dose-response curve the NR50 value is > 1000 μg/ml, therefore the MMAS is < 25.

Applicant's summary and conclusion

Interpretation of results:

other: the substance is considered to have a no/mild irritation potential.

Conclusions:

The substance is considered to have a no/mild irritation eye potential.

Executive summary:

The eye irritation potential of the substance was quantitavely assessed by the cytotoxicity assay by using the NRU (Neutral Red Uptake) assay. Fibroblasts Balb 3T3 clone 31 were exposed to the substance at eight concentrations ranging from 2000 μg/ml to 15.6 μg/ml, for 24 hours. At the end of the incubation period the medium was removed and the cells were washed with PBS and fresh culture medium and Neutral Red dye was added to the cells. The cells were incubated for 3 hours at 37 °C and 5 % CO2, after which the NR-medium was removed, the cells were washed with PBS to solubilise the NR captured by the cells. The absorbance of neutral red was measured at 540 nm. Negative controls (corn oil and culture medium) and positive control (SDS) run in parallel. The cell viability and the NR50 were calculated.

The NR50 of the sample was calculated to be > 1000 μg/ml corresponding to a MMAS < 25.

The substance is considered to have a no/mild irritation eye potential.