(1R,2S,5R)-2-isopropyl-N-(4-methoxyphenyl)-5-methylcyclohexanecarboxamide

Endpoint summary

Description of key information

Skin irritation: not irritating (OECD 404, GLP compliant)
Eye irritation: not irritating (OECD 437 (2013), GLP compliant)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-01-20 to 2009-01-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

adopted 2002-04-24

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

date of inspection 2008-08-19

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK
- Age at study initiation: twelve to twenty weeks old
- Weight at study initiation: 2.48 to 2.68 kg
- Housing: individually housed in suspended cages.
- Diet (ad libitum): 2030 Teklad Global Rabbit diet
- Water (ad libitum): mains drinking water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23 °C
- Relative humidity: 30 to 70 %
- Air exchanges: at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 g of the test material, moistened with vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 mL of distilled water

Duration of treatment / exposure:

4 hours

Observation period:

Approximately 1 hour as well as 24, 48 and 72 hours after patch removal

Number of animals:

3 rabbits

Details on study design:

TEST SITE
- Area of exposure: shaved epidermis on the dorsal/flank area
- Type of wrap used: 2.5 cm x 2.5 cm cotton gauze patch secured in position with a strip of surgical adhesive tape. The trunk of each rabbit was wrapped in an elasticated corset.

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: Four hours after application
- Washing: any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.

SCORING SYSTEM: according to the Draize scale
Any other skin reactions, if present, were also recorded.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

0

Irritant / corrosive response data:

No evidence of skin irritation was noted during the study.
No corrosive effects were noted.

Other effects:

All animals showed expected gain in body weight during the study.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance is not irritating to the skin based on an in vivo study (OECD 404). According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin irritant.

Executive summary:

The skin irritation potential of the substance was investigated according to the OECD guideline 404. A single dose of 0.5 g of the substance moistened with distilled water was applied to the intact skin of three New Zealand White rabbits and covered semi-occlusive for an exposure period of 4 hours. The test sites were examined for skin reactions and scored according to the Draize scale after 1, 24, 48 and 72 hours. Signs of skin irritation or corrosion were not observed. According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classifiication as skin irritant.

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

Study period:

2011-10-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study with minor deviations to the OECD 437 (adopted July, 26th 2013) without effects on results: - IVIS cut-off values used for identifying test chemical were different, but no influence to the classification of the test item. - information on corneal diameter and time interval prior to initiating testing with the eyes were missing

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted 2009-09-07

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or tissues and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Vehicle:

other: 0.9% (w/v) NaCl in deionised water

Controls:

other: Control (saline) bovine corneae were used.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: 10 % (w/v) suspension in vehicle

Duration of treatment / exposure:

10 minutes (± 30 seconds)

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

COLLECTION OF BOVINE EYES
- isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir and were transported in Hank's buffered salt solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature.

PREPARATION OF CORNEAE
- all eyes were carefully examined macroscopically for defects.
- the cornea was carefully removed from the eye.
- each cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both compartments of the holder were filled with complete medium.
- for equilibration, the corneae in the holder were incubated in a vertical position for one hour at 32 ± 1 °C in a water-bath.
- at the end of the incubation period, the basal opacity was determined (t0) of all cornea.
- each corneae with a value of the basal opacity > 7 was discarded.

EXPOSURE OF THE TEST GROUPS TO THE CORNEAE
- the anterior compartment received the test item or negative control or positive control (2-Ethoxyethanol; tested neat) at a volume of 0.75 mL on the surface of the corneae.
- the corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
- the test item, positive control and negative control were tested in triplicate.
- the test item or control items were rinsed off with cMEM (complete medium).
- the corneae were then incubated at 32 ± 1 °C for further two hours in a vertical position, followed by a second opacity reading (t130).
- permeability of the corneae was determined.

PERMEABILITY MEASUREMENT
- after the final opacity measurement, the complete medium was removed from the anterior compartment and replaced by 0.5 % (w/v) fluorescein solution in HBSS.
- corneae were incubated in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.
- complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm was determined with a spectrophotometer.

EVALUATION OF RESULTS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t130 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score (IVIS) of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group was calculated from the individual IVIS values.


CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if:
- in vitro irritation score of the positive control was ≥ 30
- in vitro irritation score of the negative control was ≤ 3

Irritation parameter:

other: in vitro irritancy score

Basis:

mean

Time point:

other: 10 minutes

Score:

0.55

Irritant / corrosive response data:

Relative to the negative control, the test item did not cause any increase of the corneal opacity or permeability.

Table 1: Results after 10 minutes incubation time

Test group	Opacity value = Difference (t130 – t0) of opacity		Permeability at 490 nm (OD490)		In vitro score	Mean in vitro irritation score
		Mean		Mean
Negative control	-1	-0.67	0.064	0.058	-0.04	0.21
	-1		0.055		-0.18
	0		0.056		0.84
Positive control	63.67*		0.319*		68.45	76.72
	62.67*		1.071*		78.73
	60.67*		1.489*		83.00
Test item	0.67*		-0.003*		0.62	0.55
	-0.33*		-0.012*		-0.52
	1.67*		-0.008*		1.54

* corrected values

- With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS= 0.21).

- The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae (mean IVIS =76.72).

Table 2: Historical data

	Positive control	Negative control
Mean in vitro irritation score	78.94	1.09
Standard deviation	22.76	0.97

Values of 104 studies with liquid test items performed from 2007 until 2011

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Relative to the negative control, the substance did not cause any increase of the corneal opacity or permeability. Thus, the substance is not irritating to the eye (IVIS = 0.55) under the experimental conditions. A substance with an in vitro irritation score ≤ 3 does not require classification for eye irritation and serious eye damage according to OECD 437 (2013). According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not irritating to the eye.

Executive summary:

The eye irritation potential was examined according to the OECD guideline 437 (2009) . Three fresh bovine corneae were exposed to the substance for 10 minutes, rinsed and incubated for another 120 minutes. Corneal opacity and permeability were measured and the irritancy score was calculated. Relative to the negative control, the substance did not cause any increase of the corneal opacity or permeability. The mean irritance score amounts to 0.55. According to OECD 437 (2013), a substance with an in vitro irritance score ≤ 3 does not require classification for eye irritation and serious eye damage. According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not irritating to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The substance was not observed to be irritating to the skin in a reliable in vivo skin irritation study according to OECD 404.

Eye irritation:

The substance was not oberved to be irritating to the eye in a reliable in vitro eye irritation study according to OECD 437.

Justification for selection of skin irritation / corrosion endpoint:
GLP guideline study conducted with the test item

Justification for selection of eye irritation endpoint:
GLP guideline study conducted with the test item

Justification for classification or non-classification

Skin irritation

The substance does not possess a skin irritation potential based on an in vivo OECD 404 test and does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

The substance does not possess an eye irritation potential according to an in vitro OECD 437 test and does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.