[2-(cyclohexyloxy)ethyl]benzene

Endpoint summary

Description of key information

Skin corrosion: Despite Phenafleur being a skin irritant, it is not a corrosive because the substance does not contain corrosive functional groups (acids, bases or highly reactives). This is further confirmed as the substance is not an eye irritant.

Skin irritation (OECD TG 439): Irritating
Eye irritation (OECD TG 405): The substance is not an ocular irritant.

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

Study period:

11 April, 2016 - 18 April, 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:

(2015)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2012)

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-015
- The solid test item was melted before use and twenty five μL of the undiluted test substance (liquid) 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: 35.1 - 36.5°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 thecellular 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:
Phenafleur 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 Phenafleur 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.
DATA EVALUATION:
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:

other: other: relative mean viability

Value:

34

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

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

Other effects / acceptance of results:

Direct MTT Reduction:
Phenafleur 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 Phenafleur 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 Phenafleur compared to the negative control tissues was 34%. Since the mean relative tissue viability for Phenafleur was below 50% Phenafleur is considered to be irritant.

Quality Criteria:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 25%. The positive control meets the validity criterionwas within the laboratory historical control data range. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The SD calculated from individual % tissue viabilities of the three identically treated replicates of the negative control was 19%.
Evaluation: The SD calculated from individual % tissue viabilities of the three identically treated replicates of the negative control was just above the acceptability criteria of 18% (18.68%). The individual viabilities were all three clearly negative and additionally had an OD which was within the historical data range. Therefore this deviation has no influence on the study integrity.

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

Item	OD570 of tissues	Mean OD562 of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)
Negative Control Item	0.908	0.935	0.176		100
	0.774
	1.123
Positive Control Item	0.221	0.248	0.165	24	25
	0.097			13
	0.425			38
Test Item	0.387	0.319	0.078	43	34
	0.235			30
	0.335			30

SD = Standard deviation

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

Interpretation of results:

other: Skin irritant Cat. 2

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 substance compared to the negative control tissues was 34%. Since the mean relative tissue viability for the substance was below 50%, it is considered to be a skin 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 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 25% after 15 minutes exposure. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 34%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment, the substance is considered to be a skin irritant.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 - 24 March 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The present in vivo study can be used because it was performed before the current REACH requirements on in vitro testing (October, 2016)

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Remarks:

albino strain

Details on test animals or tissues and environmental conditions:

- Source: Perfection Breeders, Inc., Douglassville, Pennsylvania
- Age at study initiation: No information provided
- Weight at study initiation: Body weights were in range of 2-3 kg
- Housing: Individually
- Diet: Wayne Rabbit Ration, ad libitum, checked daily and added or replaced as needed, checked for contaminants.
- Water: Fresh tap water, available ad libitum, checked for contaminants.
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS: The light cycle was: 12 hours light, 12 hours dark. The temperature was attempted to be maintained at the level of 20 ± 3°C and the humidity in the range of 30-70%. The waste material was removed daily. Cages and feeders were sanitized every two weeks.

Vehicle:

unchanged (no vehicle)

Controls:

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

Amount / concentration applied:

Amount applied (volume or weight with unit): 0.1 mL

Duration of treatment / exposure:

Single instillation on Day 1

Observation period (in vivo):

7 days

Number of animals or in vitro replicates:

3 males and 3 females

Details on study design:

STUDY DESIGN
The test material was applied at 100% in 6 animals (3 males and 3 females).

TREATMENT
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 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 (mean)

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal: #1, 2, 3, 4, 5 and 6 (mean)

Time point:

7 d

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal: #1, 2, 3, 4, 5 and 6 (mean)

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal: #1, 3, 4, 5 and 6 (mean)

Remarks:

(mean)

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #2

Remarks:

(mean)

Time point:

24/48/72 h

Score:

0.33

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

animal: #1, 2, 3, 4, 5 and 6 (mean)

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal: #1, 2, 3, 4, 5 and 6 (mean)

Time point:

7 d

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal: #1, 2, 3, 4, 5 and 6 (mean)

Time point:

7 d

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal: #1, 2, 3, 4, 5 and 6 (mean)

Time point:

7 d

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal: #1, 2, 3, 4, 5 and 6 (mean)

Time point:

other: 1 hr

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal: #1, 2, 3, 4, 5 and 6 (mean)

Time point:

other: 1 hr

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal: #1, 2, 3, 4, 5 and 6 (mean)

Time point:

other: 1 hr

Score:

0.83

Max. score:

1

Reversibility:

fully reversible within: 48 hr

Irritation parameter:

chemosis score

Basis:

animal: #1, 2, 3, 4, 5 and 6 (mean)

Time point:

other: 1 hr

Score:

0.17

Max. score:

1

Reversibility:

fully reversible within: 24 hr

Rabbit no.	Observations	1 hr	24 hr	48 hr	72 hr	7 days
1301	Cornea	0,0=0	0,0=0	0,0=0	0,0=0	0,0=0
	Iris	0=0	0=0	0=0	0=0	0=0
	Conjunctiva	1,0,0=2	0,0,0=0	0,0,0=0	0,0,0=0	0,0,0=0
1302	Cornea	0,0=0	0,0=0	0,0=0	0,0=0	0,0=0
	Iris	0=0	0=0	0=0	0=0	0=0
	Conjunctiva	1,0,0=2	1,0,0=2	0,0,0=0	0,0,0=0	0,0,0=0
1303	Cornea	0,0=0	0,0=0	0,0=0	0,0=0	0,0=0
	Iris	0=0	0=0	0=0	0=0	0=0
	Conjunctiva	0,0,0=0	0,0,0=0	0,0,0=0	0,0,0=0	0,0,0=0
1304	Cornea	0,0=0	0,0=0	0,0=0	0,0=0	0,0=0
	Iris	0=0	0=0	0=0	0=0	0=0
	Conjunctiva	1,0,0=2	0,0,0=0	0,0,0=0	0,0,0=0	0,0,0=0
1305	Cornea	0,0=0	0,0=0	0,0=0	0,0=0	0,0=0
	Iris	0=0	0=0	0=0	0=0	0=0
	Conjunctiva	1,0,0=2	0,0,0=0	0,0,0=0	0,0,0=0	0,0,0=0
1306	Cornea	0,0=0	0,0=0	0,0=0	0,0=0	0,0=0
	Iris	0=0	0=0	0=0	0=0	0=0
	Conjunctiva	1,1,0=4	0,0,0=0	0,0,0=0	0,0,0=0	0,0,0=0

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 according to OECD 405 (1981), no irritation was observed. Based on the results of this study, the substance is not considered an eye irritant.

Executive summary:

The substance was tested in an eye irritation test in rabbits according to OECD 405 (1981). Six New Zealand albino rabbits were used (3 males and 3 females). The volume applied was 0.1 ml. Ocular effects included only a slight redness of conjunctiva in 5 out of 6 animals (score 1) at 1 hr, which were fully reversible within 24 hours in 4 animals and within 48 hours in 1 animal. A slight chemosis was observed in 1 animal (score 1) that was fully resolved within 24 hours. No other effects were seen, resulting in absence of eye irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin corrosion:

Despite Phenafleur being a skin irritant, it is not a corrosive because the substance does not contain corrosive functional groups (acids, bases or highly reactives). This is further confirmed as the substance is not an eye irritant.

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 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 25% after 15 minutes exposure. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 34%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment, the substance is considered to be a skin irritant.

Eye irritation OECD TG 405:

The substance was tested in an eye irritation test in rabbits according to OECD 405 (1981). Six New Zealand albino rabbits were used (3 males and 3 females). The volume applied was 0.1 ml. Ocular effects included only a slight redness of conjunctiva in 5 out of 6 animals (score 1) at 1 hr, which were fully reversible within 24 hours in 4 animals and within 48 hours in 1 animal. A slight chemosis was observed in 1 animal (score 1) that was fully resolved within 24 hours. No other effects were seen, resulting in absence of eye irritation.

Justification for classification or non-classification

Based on the positive results in the skin irritation test, the substance needs to be classified as a skin irritant category 2 and shall be labelled as H315: Causes skin irritation according to Regulation (EC) No. 1272/2008 (and its amendments); Based on the negative results in the eye irritation test, the substance does not need to be classified for this endpoint according to EU CLP regulation (1272/2008) and its amendments.