4-(isopropyl)cyclohexyl propionate

Endpoint summary

Description of key information

Skin corrosion: The substance is not a skin or eye irritant, therefore the substance is also not a skin corrosive.

Skin irritation (OECD TG 439): Not irritating
Eye irritation and severe damage (OECD TG 405): Not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 August 2016 - 29 August 2016

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)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number: 16-EKIN-034
- Twenty five μL of the undiluted test substance was added into 12-well plates on top of the skin tissues.
- The test item was applied topically to the corresponding tissues ensuring uniform covering.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 35.3 - 37.1°C

PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.
Test for Direct MTT Reduction + colour interference:
The test substance was checked for possible direct MTT reduction and colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the exposure. To assess the colour interference, 10 μL of the test substance was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed. To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.

PRE-INCUBATION:
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 22 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.
APPLICATION/TREATMENT OF TEST SUBSTANCE:
The test was performed on a total of 3 tissues per test item together with negative and positive controls.
Twenty five μL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.
CELL VIABILITY MEASUREMENT:
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.
DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test material
- Applied volume: 25 μL

Duration of treatment / exposure:

15-Minute exposure period and 42 hours post-exposure incubation period.

Number of replicates:

A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Irritation / corrosion parameter:

% tissue viability

Value:

123

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The relative mean tissue viability compared to the negative control tissues (100%).

Other effects / acceptance of results:

Direct MTT Reduction:
The test item was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that the test item did not interact with the MTT endpoint.

Test Item, Positive Control Item and Negative Control Item
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 123%. Since the mean relative tissue viability for Veilex 4 was above 50% it is considered to be non-irritant.

Quality Criteria
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 11%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage
viability of three tissues treated identically was 7% or less, indicating that the test system functioned properly.

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item	OD570 of tissues	Mean OD570 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)
Negative Control Item	0.746	0.744	0.052		100
	0.691
	0.794
Positive Control Item	0.061	0.083	0.025	8	11
	0.076			11
	0.110			14
Test Item	0.920	0.917	0.035	123	123
	0.952			138
	0.881			111

SD = Standard deviation

*The mean viability of the negative control tissues is set at 100 %

Interpretation of results:

other: Not a skin irritant.

Remarks:

According to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 123%. Since the mean relative tissue viability for the test substance was above 50% it is considered to be non-irritant.

Executive summary:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours post incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 11% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 7%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 123%. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment the substance is considered to be non-irritant.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation, other

Remarks:

Test performed before current guidelines

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted in March 1977.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Reliability 2 is assigned because although the study was conducted similar to the OECD TG 405, the guideline is not referenced and there is no documentation on experimental conditions. The study is also non-GLP. However, this did not influence the reliability of the results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Remarks:

albino strain

Vehicle:

unchanged (no vehicle)

Controls:

other: One eye of each animal remained untreated and served as the reference control.

Amount / concentration applied:

Amount applied: 0.1 mL. Undiluted

Duration of treatment / exposure:

Single instillation on Day 1, exposure for 24 hours

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

6 animals

Details on study design:

STUDY DESIGN
The test material was applied at 100% in 6 animals.

TREATMENT
Similar and in accordance with OECD 405 (1981). The test substance is placed in the conjunctival sac of one eye of each animal after gently pulling the lower lid away from the eyeball. The lids are then gently held together for about one second. The other eye serves at control.

REMOVAL OF TEST SUBSTANCE
-Washing: No

OBSERVATIONS
- Irritation:
The eyes of each animal were examined at 1, 24, 48 and 72 hours and 5 and 7 days after instillation of the test substance.
The irritation scores and a description of all other (local) effects were recorded. The irritation was assessed according to OECD 405 (1981).

Irritation parameter:

cornea opacity score

Basis:

animal: #1, 2, 3, 4, 5 and 6

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal: #1, 2, 3, 4, 5 and 6

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal: #1 and 3

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 24 h

Irritation parameter:

conjunctivae score

Basis:

animal: #2, 4, 5 and 6

Time point:

24/48/72 h

Score:

0

Max. score:

0

Results:

Rabbit no.	Observations	1 hr	24 hr	48 hr	72 hr	5 days	7 days
1	Cornea	0	0	0	0	0	0
	Iris	0	0	0	0	0	0
	Conjunctiva	6	4	0	0	0	0
2	Cornea	0	0	0	0	0	0
	Iris	0	0	0	0	0	0
	Conjunctiva	6	0	0	0	0	0
3	Cornea	0	0	0	0	0	0
	Iris	0	0	0	0	0	0
	Conjunctiva	6	4	0	0	0	0
4	Cornea	0	0	0	0	0	0
	Iris	0	0	0	0	0	0
	Conjunctiva	6	0	0	0	0	0
5	Cornea	0	0	0	0	0	0
	Iris	0	0	0	0	0	0
	Conjunctiva	6	0	0	0	0	0
6	Cornea	0	0	0	0	0	0
	Iris	0	0	0	0	0	0
	Conjunctiva	6	0	0	0	0	0

4 - Slight erythema, slight discharge (Draize score = 1)

6 - Slight erythema, moderate discharge (Draize score = 1)

The scores in the table above are from F.H.S.A (a US test guideline) and are different from Draize scores. The wording is therefore used qualitatively to convert the F.H.S.A scores into Draize scores. Slight erythema is considered to be Draize 1.

Interpretation of results:

other: Not an eye irritant

Remarks:

According to EU CLP 1272/2008 and its amendments.

Conclusions:

In an eye irritation study with rabbits, performed similar to OECD 405 (1981), essentially no irritation was observed. Based on the results of this study, test substance is not an eye irritant.

Executive summary:

The substance was tested in an eye irritation test in rabbits similar to OECD 405 (1981). Six New Zealand albino rabbits were used. The volume applied was 0.1 ml and the material was applied undiluted. The scores in the test are from F.H.S.A (a US test guideline) and they are different from Draize scores. The wording is therefore used qualitatively to convert the F.H.S.A scores into Draize. Slight erythema and moderate discharge were seen in all animals at 1 hour, slight erythema and slight discharge were seen in 2/6 animals at 24 hours and none was seen after 48 hours. Discharge is not an endpoint for classification and labelling. Slight erythema is considered to be Draize 1. The mean conjunctiva score in the two affected animals was 0.3, based on the time points of 24, 48 and 72 hours, which is smaller than 2. Based on the results of this study, test substance is not an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours post incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 11% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 7%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 123%. Since the mean relative tissue viability for the substance was above 50% after 15 minutes treatment the substance is considered to be non-irritant.

Eye irritation OECD TG 405:

The substance was tested in an eye irritation test in rabbits similar to OECD 405 (1981). Six New Zealand albino rabbits were used. The volume applied was 0.1 ml and the material was applied undiluted. The scores in the test are from F.H.S.A (a US test guideline) and they are different from Draize scores. The wording is therefore used qualitatively to convert the F.H.S.A scores into Draize. Slight erythema and moderate discharge were seen in all animals at 1 hour, slight erythema and slight discharge were seen in 2/6 animals at 24 hours and none was seen after 48 hours. Discharge is not an endpoint for classification and labelling. Slight erythema is considered to be Draize 1. The mean conjunctiva score in the two affected animals was 0.3, based on the time points of 24, 48 and 72 hours, which is smaller than 2. Based on the results of this study, test substance is not an eye irritant.

Justification for classification or non-classification

Based on the negative results in the skin and eye irritation tests, the substance does not need to be classified for this endpoint in accordance with EU CLP regulation (1272/2008) and its amendments.