1-Butoxy-4-[4-(trans-4-ethylcyclohexyl)-1-cyclohexen-1-yl]-2,3-difluorobenzene

Endpoint summary

Description of key information

Skin irritation

OECD 439: negative

OECD 404: negative

Eye irritation

HET-CAM: negative

OECD 405 negative

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:

weight of evidence

Study period:

Apr 25, 2017 - June 14, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Council Regulation (EC) No. 761/2009 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No pre-treatment; the test item was applied undiluted.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

standard model

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used in this study; The test item was applied neat to the tissues.

Details on test system:

CELL CULTURE
- Supplier:SkinEthic Laboratories (Nice, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch No: 17-RHE-050
- Expiration date: May 8, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL PBS using a pipette. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL: 16 mg of solid test material
NEGATIVE CONTROL: 16 µL (Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)

Duration of treatment / exposure:

42 min (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

87.9

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
After treatment with the negative control the absorbance values reached a mean OD of 1.871 (SD 9.0%). Therefore, the negative control fulfilled the validity criteria.
Treatment with the positive control revealed a mean relative viability of 1.8% (SD 16.7%), thus the positive control reached the validity criteria.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.871	100
Positive Control	42	0.034	1.8
Test Material	42	1.645	87.9

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439.Under the experimental conditions reported, the test material is not irritating to the skin.

Executive summary:

Objective

This in vitro study was performed to assess the irritation potential of the test item by means of the Reconstructed Human Epidermis Test.

Study Design

The test consisted of a topical exposure of the test material to a human reconstructed model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential.Triplicates of the human skin model RHEä(Reconstructed Human Epidermis, SkinEthic) were treated with the test material, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (PBS-buffer) or the positive control (aqueous solution of sodium dodecyl sulfate) were applied to each tissue. Before adding the solid test material, 10 µL of deionised water was spread to the epidermis surface to improve further contact between the test material and the epidermis. Afterwards, 16 mg of the test material were applied to each tissue.

Results

After treatment with the negative control (DPBS-buffer) the mean OD was 1.871. Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.8%. Thus, the acceptance criteria stated in part 5.9 were met.

Following treatment with the test item, the tissue viability was 87.9% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Conclusion

Under the experimental conditions reported, the test material is not irritating to the skin.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 Feb 2020 - 18 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed for a registration in a non-EU country.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Species: Rabbit
Strain: New Zealand White
Condition: SPF-Quality
Source: Charles River France, L’Arbresle, France
Number of Animals: 3 Males
Age at the Initiation of Dosing: A young adult animal (approximately 11-13 weeks old) was selected.
Weight at the Initiation of Dosing: 2246 to 2313 g
Acclimatisation: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 °C
- Humidity (%): 53 to 55%.
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12/12 hours

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 g

Duration of treatment / exposure:

4 h

Observation period:

14 days

Number of animals:

1 female

Details on study design:

TEST SITE
Approximately 24 hours before dosing, the dorsal fur was clipped with an electric clipper, exposing an area of approximately 150 square centimeters (10x15 cm). To facilitate scoring,
the treated skin area was re-clipped at least 3 hours before the observations.
The animal was treated by dermal application of 0.5 grams of the test item. The test item was applied to the skin of one flank, using a metalline patch of 2x3 cm. The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage. Four hours after the application, the dressing was removed and the skin cleaned of residual test item using tap water.

SCORING SYSTEM:
- Method of calculation:

The skin reactions were assessed at approximately 1, 24, 48 and 72 hours and 7 and 14 days after the removal of the dressings and test item. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of the untreated skin of the animal served as control.
The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

Erythema and eschar formation:
No erythema .............................................................................................................................................................. 0
Very slight erythema (barely perceptible) ................................................................................................................. 1
Well-defined erythema ............................................................................................................................................... 2
Moderate to severe erythema..................................................................................................................................... 3
Severe erythema (beef redness) * ............................................................................................................................ 4
*. Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for erythema (= 4) is given.

Oedema formation:
No oedema.................................................................................................................................................................. 0
Very slight oedema (barely perceptible)..................................................................................................................... 1
Slight oedema (edges of area well-defined by definite raising)................................................................................. 2
Moderate oedema (raised approximately 1 millimeter)................................................................................................ 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) ................................. 4

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

No skin irritation was caused by 4 hours exposure to the test material.

Other effects:

No signs of systemic toxicity were observed in the animal during the test period and no mortality occurred.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results the test material does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this primary skin irritation study was to assess the possible irritation or corrosion potential of a single dose of the test material when administered to the intact skin of rabbits.

The study was carried out in compliance with the guidelines described in:

• OECD No. 404 (2015) "Acute Dermal Irritation / Corrosion".

• EC No 440/2008, part B: "Acute Toxicity: Dermal Irritation/Corrosion".

• EPA, OPPTS 870.2500 (1998), "Acute Dermal Irritation".

• JMAFF Guidelines (2000), including the most recent revisions.

Three rabbits were exposed to 0.5 grams of the test material, moistened with 0.3 mL 50% watery ethanol by application onto clipped skin for 4 hours using a semi-occlusive

dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure.

No skin irritation was caused by 4 hours exposure to the test material.

Based on these results the test material does not have to be classified and has no obligatory labelling requirement for skin irritation according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Mar 03 2008 - Apr 17 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: Hen's egg test on the chorioallantoic membrane (HET-CAM) for irritating potential

Principles of method if other than guideline:

During the second part of the 80' s, with a growing number of chemicals to be tested and due to an increasing awareness towards the need to protect animals from painful experiments, the hen's egg chorioallantoic membrane test (HET-CAM) was developed. The chorioallantoic membrane (CAM) of fertilized and incubated hen eggs provides a suitable model for membranes with an intact and functional vascular system. The CAM has no nerve fibers in the early stages of development and is regarded as being insensitive to pain. For these reasons, the HET-CAM is believed to be a reliable alternative to the primary eye irritation test in rabbits, which does not conflict with ethical and egal aspects of animal protection.

GLP compliance:

yes (incl. QA statement)

Species:

other: hen's eggs, fertilized

Strain:

other: White leghorn

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
-Source: LSL Rhein Main Geflügelvermehrungsbetrieb, Dieburg, Germany

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

Amount / concentration applied:

The tip of a spatula of the test item was applied on the vascular chorioallantoic membrane (CAM). The application volume of the reference material was 0.3 mL.

Duration of treatment / exposure:

Only eggs without injuries of the membrane or other abnormalities were used for testing. After application of the test material, the blood vessels, including the capillary system, and the albumin were examined and scored for irritating effects (haemorrhage, lysis, and coagulation) over a period of 300 sec. The period between treatment and the onset of the reactions was measured in seconds and recorded with stopwatch and protocol.

Number of animals or in vitro replicates:

6 eggs were used

Details on study design:

The irritating potential of the test item was determined in the hen's egg test on the chorioallantoic membrane (HET-CAM). Fertile hen eggs on day 10 of incubation provided the test system. The eggshell was removed around the air cell by means of a dentist drill and the shell with attached outer membrane and inner egg membrane was carefully opened. After the membrane had been moistened with isotonic sodium chloride solution, it was removed to expose the chorioallantoic membrane (CAM) of the fertilized eggs, which lies immediately below. After application of the test material, the blood vessels, including the capillary system, and the albumin were examined and scored for irritating effects (haemorrhage, lysis, and coagulation) over a period of 300 sec. As reference material Dodecyl sulfate sodium salt was tested and evaluated to ensure the sensitivity of the system.

Scoring system according to: Lüpke, NP (1985) Hen's egg chorioallantoic membrane test for irritation potential. Fd. Chem. Toxic. 23 (2), 287-291.
Cumulative score Assessment of irritancy
0 – 0.9 None
1 – 4.9 Mild
5 - 8.9 Moderate
9 - 21 Severe

Irritation parameter:

in vitro irritation score

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

Score: 9.69

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given experimental conditions the test item showed no irritating effects in the HET-CAM test.

Executive summary:

Study design

The irritating potential of the test item was determined in the hen's egg test on the chorioallantoic membrane (HET-CAM). This test is routinely used as a screening assay prior to in vivo tests in rabbits. Fertile hen eggs on day 10 of incubation provided the test system. The egg shell was removed around the air cell by means of a dentist drill. The shell with attached outer membrane and inner egg membrane was carefully opened. After the membrane had been moistened with isotonic sodium chloride solution, it was removed to expose the chorioallantoic membrane (CAM) of the fertilized eggs, which lies immediately below. After application of the test material, the blood vessels, including the capillary system, and the albumin were examined and scored for irritating effects (haemorrhage, lysis, and coagulation) over a period of 300 sec. As reference material Dodecyl sulfate sodium salt was tested and evaluated to ensure the sensitivity of the system.

Results

For the test item a cumulative score of 0 (no irritancy) was obtained, while dodecyl sulfate sodium salt as reference material gave a score of 10.59 (severe irritancy).

Conclusion

Under the given experimental conditions, the test item showed no irritating effects in the HET-CAM test.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Mar 06 - May 26, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed for a registration in a non-EU country.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Age at study initiation: approximately 12-13 weeks old
- Weight at study initiation: 2372 to 2813 g.
- Housing: ndividually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19
- Humidity (%): 52 *- 55
- Air changes (per hr): >10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.05 mg (range 0.0509-0.0511 mg) of the test item (a volume of approximately 0.1 mL)

Duration of treatment / exposure:

72 h

Observation period (in vivo):

1, 24, 48, 72 h

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 1 and 24 hour

SCORING SYSTEM:

The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

CORNEAL IRRITATION
Opacity: degree of density (area most dense taken for reading)
No ulceration or opacity (may include slight dulling of normal luster) .0
Scattered or diffuse areas of opacity, details of iris clearly visible .1
Easily discernible translucent area, details of iris slightly obscured .2
Nacreous area, no details of iris visible, size of pupil barely discernible .3
Opaque cornea, iris not discernible through the opacity .4
Area of cornea involved:
No ulceration or opacity .0
One quarter or less but not zero .1
Greater than one quarter, but less than half .2
Greater than half, but less than three quarters .3
Greater than three quarters, up to whole area .4
 
IRIS
Normal .0
Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia, or injection, any of
these or combination thereof, iris still reacting to light (sluggish reaction is positive) .1
No reaction to light, hemorrhage, gross destruction (any or all of these) .2
CONJUNCTIVAL IRRITATION
Redness (refers to palpebrae and sclera, excluding cornea and iris):
Blood vessels normal .0
Some blood vessels definitely hyperaemic (injected) .1
Diffuse, crimson color, individual vessels not easily discernible .2
Diffuse beefy red .3
Chemosis (refers to lids and/or nictitating membranes):
No swelling ………………………………………… .0
Any swelling above normal (includes nictitating membranes) .1
Obvious swelling with partial eversion of lids .2
Swelling with lids about half closed .3
Swelling with lids more than half closed .4
Discharge:
No discharge (may include small amounts observed in inner canthus of normal animals) 0
Any amount different from normal and/or lacrimation 1
Discharge with moistening of the lids and hairs just adjacent to lids 2
Discharge with moistening of the lids and hairs (considerable area around the eye) 3

Where standard lighting was considered inadequate for observing minor effects, eye examinations were performed using an ophthalmic examination lamp.


TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

0

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.3

Max. score:

1

Reversibility:

fully reversible within: 48 hours

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Instillation of approximately 0.05 mg of the test material (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in irritation of the
conjunctivae, which consisted of redness and discharge in all animals and chemosis in one animal only. The irritation had completely resolved within 24 hours in one animal and within 48 hours in the other two animals.
No iridial irritation or corneal opacity was observed and treatment of the eyes with 2% fluorescein 24 hours after test item instillation revealed no corneal epithelial damage.

Other effects:

Remnants of the test item were present in the eye at one hour after instillation in all animals.
No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Individual Eye Irritation Scores

Animal	Timepoint after Dosing	Cornea			Iris	Conjunctivae			Comments
		Opacity (0-4)	Area (0-4)	Fluor Area / [%]^2	(0-2)	Redness (0-4)	Chemosis (0-4)	Discharge (0-4)
494^1	1 hour	0	0	n.a.	0	1	0	1	b
	24 hours	0	0	0	0	1	0	0	-
	48 hours	0	0	n.a.	0	0	0	0	-
	72 hours	0	0	n.a.	0	0	0	0	-
504	1 hour	0	0	n.a.	0	1	1	1	b
	24 hours	0	0	0	0	1	0	0	-
	48 hours	0	0	n.a.	0	0	0	0	-
	72 hours	0	0	n.a.	0	0	0	0	-
507	1 hour	0	0	n.a.	0	1	0	1	b
	24 hours	0	0	0	0	0	0	0	-
	48 hours	0	0	n.a.	0	0	0	0	-
	72 hours	0	0	n.a.	0	0	0	0	-

1 Sentinel
²Green staining after fluorescein treatment (% of total corneal area) indicating corneal epithelial damage
n.a. not applicable
b  Remnants of the test item in the eye
-  No comments

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results, the test material does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized
System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling
and packaging of items and mixtures (including all amendments).

Executive summary:

The objective of this acute eye irritation study was to assess the possible irritation or corrosion potential when a single dose of the test material was placed in the

conjunctival sac of the rabbit eye.

The study was carried out in compliance with the guidelines described in:

• OECD No.405 (2017) "Acute Eye Irritation / Corrosion".

• EC No 440/2008, part B: "Acute Toxicity: Eye Irritation/Corrosion".

• EPA, OPPTS 870.2400 (1998), "Acute Eye Irritation".

• JMAFF Guidelines (2000), including the most recent revisions.

Single samples of approximately 0.05 mg of the test material (a volume of approximately 0.1 mL) were instilled into one eye of each of three rabbits. Observations were

made 1, 24, 48 and 72 hours after instillation. Instillation of the test item resulted in irritation of the conjunctivae, which consisted of redness and discharge in all animals and chemosis in one animal only. The irritation had completely resolved within 24 hours in one animal and within 48 hours in the other two animals. No iridial irritation or corneal opacity was observed and treatment of the eyes with 2% fluorescein 24 hours after test item instillation revealed no corneal epithelial damage.

Remnants of the test item were present in the eye at one hour after instillation in all animals.

Based on these results, the test material does not have to be classified and has no obligatory labelling requirement for eye irritation according to the Globally Harmonized

System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling

and packaging of items and mixtures (including all amendments).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided information there is no need to classified according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.