Reaction mass of (1RS,2RS,7SR,8RS,9E)-9-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane and (1RS,2RS,7SR,8RS,9Z)-9-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane and (1RS,2SR,7SR,8SR,10E)-10-Ethylidene-3-oxatricyclo[6.2.1.0(2,7)] undecane and (1RS,2SR,7SR,8SR,10Z)-10-Ethylidene-3-oxatricyclo [6.2.1.0(2,7)] undecane

Endpoint summary

Description of key information

Skin irritation: non-irritating, HRIPT, Wilson 1981
Skin irritation: non-irritating, OECD 404, Regan 1981
Eye irritation: non-irritating, OECD 405, Teunissen 2013

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF including guidelines (2011); the most recent partial revisions.

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: 12 - 24 weeks old
- Weight at study initiation: at least 1.5kg
- Housing: Animals were individually housed in labeled cages with perforated floors and shelters.
- Diet: pelleted diet and wooden sticks ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod: 12 hour light / 12 hour dark

IN-LIFE DATES: From:22 April 2013 To:02 May 2013

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL
- Concentration (if solution): undiluted

Duration of treatment / exposure:

The test material was administered to the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance. Eyes were not washed post administration of the test material. Immediately after the 24-hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage.

Observation period (in vivo):

72 hours. (The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance).

Number of animals or in vitro replicates:

3 males

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

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


TOOL USED TO ASSESS SCORE: ophthalmic examination lamp.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0.7

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48 and 72 h

Score:

0.3

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

In all three animals, irritation of the conjunctivae, which consisted of redness, chemosis and discharge was observed. The irritation had completely resolved within 72 hours in all animals. No iridial irritation or corneal opacity were observed, and treatment of the eyes with 2% fluorescein 24 hours after test substance instillation revealed no corneal epithelial damage.

Table1: Individual eye irritation scores

		Cornea			Iris	Conjunctivae
Organism	Time after dosing	Opacity (0-4)	Area (0-4)	Fluor area (%) #2	(0-2)	Redness (0-3)	Chemosis (0-4)	Discharge (0-3)
473 #1	1 hour	0	0	-	0	1	2	2
	24 hours	0	0	0	0	1	1	1
	48 hours	0	0	-	0	1	0	0
	72 hours	0	0	-	0	0	0	0
479	1 hour	0	0	-	0	2	1	1
	24 hours	0	0	0	0	1	1	1
	48 hours	0	0	-	0	1	0	0
	72 hours	0	0	-	0	0	0	0
480	1 hour	0	0	-	0	2	1	2
	24 hours	0	0	0	0	1	1	1
	48 hours	0	0	-	0	1	0	0
	72 hours	0	0	-	0	0	0	0

 #1         Sentinel

#2          Green staining after fluorescein treatment (percentage of total corneal area)

 

Table2: Mean value eye irritation scores

Organism	Mean 24, 48 and 72 hours
	Corneal		Iris		Conjunctivae
	opacity				Redness		Chemosis
473	0.0		0.0		0.7		0.3
479	0.0		0.0		0.7		0.3
480	0.0		0.0		0.7		0.3

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test substance is not irritating.

Executive summary:

The study was performed to assess the irritancy potential of the test material to the eye following a single application in the New Zealand White rabbit. The study was performed to GLP and the method was designed to meet the requirements of OECD Guidelines for the Testing of Chemicals No. 405. A volume of 0.1 ml of the test material was placed into the conjunctival sac of one eye of 3 animals. The other eye remained untreated and was used for control purposes. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. No corneal (opacity) or iridial effects were noted during the study. Minimal to moderate (grade 1 to 2) conjunctival irritation was noted for all animals at the 1 to 48 -hour observations.The irritation had completely resolved within 72 hours in all animals. Under the conditions of this study the test material is not considered to be irritating to New Zealand White rabbit eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

In vitro, OECD 439 2013 - The study was performed to assess the skin irritation potential of the test material to a human three dimensional epidermal model. The study was performed under GLP and followed the OECD guideline 439. Tissues were treated with the test material for 15 minutes and then washed with phosphate buffered saline to remove residual test material. Subsequently the skin tissues were incubated for 43 hours at 37°C. 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-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. The relative mean tissue viability obtained after 15 minutes treatment with the test substance compared to the negative control tissues was 10%. Since the mean relative tissue viability for the test substance was below or equal to 50% after 15 minutes treatment it is considered to be irritant.

 

In vivo, equiv. OECD 404 1981 -The study was performed under GLP and followed a method similar to OECD guideline 404. A volume of 0.5 ml of the test material was applied to each of 2 sites per rabbit. Prior to test article application, the test site on the right dorsal side was abraded. Following application, each test site was occluded with a one-inch square gauze patch. The trunk of each animal was then wrapped with an occlusive dressing. At the 24h observation period, all animals developed very slight to moderate erythema; by the 72h observation period these effects had fully reversed. An overall mean primary irritation score was determined as 0.75. Under the conditions of this study, the authors report that the test material is slightly irritating to the skin of New Zealand White rabbits. However according to the CLP classification system, the test material is not considered to be irritating to skin.

 

In vivo, equiv. OECD 402 1981 – The study was performed under GLP and followed a method similar to OECD guideline 402. A dose of 5 g/kg of test material was applied to the prepared abdomen site of each rabbit. Prior to test article application, the test site of two animals was abraded. Following application, each test site was occluded with a gauze patch. The torso of each animal was then wrapped with an occlusive dressing. At 24 hours the patches were removed and the site was wiped. One rabbit died at 24h. There were no pre-death toxic signs or abnormalities at necropsy. On 24h two animals developed minor erythema and all animals developed slight oedema. On day 7, all animals developed severe erythema and slight oedema. By day 14, only 1 animal had severe erythema and slight erythema. According to the CLP classification system, the test material would be considered to be irritating to skin.

 

Human HRIPT 1981 – The study was performed to assess the irritating and skin sensitisation potential of the test material in human volunteers. The study was not performed to GLP. The test involved the application of the test material to the upper arms of a group of 50 volunteer subjects. The test site was covered with semi-occlusive dressing and remained in place for 24 hours after which the excess test material was wiped off. The subjects were rested for a 24 hour period after which the skin site was again examined and the experimental material was again applied to the same site as previously used. The second application was identical to the first and remained in place 24 hours. This procedure was repeated 3 times a week for 3 weeks for a total of 9 applications. A 2 week rest period then elapsed after which a challenge application was applied in the same manner to the same skin site as well as to a previously untreated skin site on the same arm. The challenge applications were removed after 24hours and the two sets of test sites were examined for signs of irritation or sensitization. The sites were re-examined after 48 and 72 hours. No irritation or sensitisation effects were observed in any test subject at any time-point - in either those who were challenged through the original test site or new (previously untreated) test site. No effects were observed in the subset of test subjects where the test site was irradiated. The test material is not considered to be irritating under the conditions of this test.

 

Skin corrosion:

In vitro, OECD 431 2013 - The study was performed to assess the skin corrosion potential of the test material to a human three dimensional epidermal model. The study was performed under GLP and followed the OECD guideline 431. Tissues were treated in duplicate with the test material for 3 minutes and 1 hour and then washed with phosphate buffered saline to remove residual test material. Cell cultures were then transferred to MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate. Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 88% and 73% respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive.

 

Eye irritation/corrosion:

In vivo OECD 405 2013 - The study was performed to conclude on the classification and irritancy potential of the test material to the eye following a single application in the New Zealand White rabbit. The study was performed to GLP and the method was designed to meet the requirements of OECD Guidelines for the Testing of Chemicals No. 405. Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment. No corneal (opacity) or iridial effects were noted during the study. Minimal to moderate (grade 1 to 2) conjunctival irritation was noted for all animals at the 1 to 48 -hour observations. The irritation had completely resolved within 72 hours in all animals. Under the conditions of this study the test material is not considered to be irritating to New Zealand White rabbit eyes.

 

In vitro OCD 437 2013 - The study was performed to assess the eye irritancy potential of the test material in isolated bovine corneas. The study was performed under GLP and followed a method equivalent to OECD guideline 437. The ocular irritancy of the test substance was tested through topical application for 10 ± 1 minutes. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride, was 136 and was within the historical positive control data range. The test substance did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.9 after 10 minutes of treatment. Under the conditions of this study the test material is not considered to be a severe irritant or corrosive in the Bovine Corneal Opacity and Permeability test.

 

OECD 405 1981 - The study was performed to assess the irritancy potential of the test material to the eye following a single application in the New Zealand White rabbit. The study was performed to GLP and followed a guideline similar to OECD 405. There was limited test material information available in this study and it has a reliability score that cannot be assigned. Assessment of ocular damage/irritation was made at 24, 48 and 72 hours following treatment. No corneal (opacity) effects were noted during the study and mild iridial effects were observed in rabbit number 6 at 24h but this resolved by 48h. Minimal (grade 1) conjunctival irritation was noted in 5 rabbits at the 1 to 48 -hour observations. The irritation had completely resolved within 72 hours in all animals. According to the author, under the conditions of this study the test material is not considered to be minimally irritating to New Zealand White rabbit eyes. However when comparing the data against Regulation (EC) 1272/2008 CLP criteria, the test material is not considered to be irritating to the eyes of New Zealand white rabbits.

Justification for selection of skin irritation / corrosion endpoint:
Five studies available, two in vitro GLP compliant Klimisch 1; two in vivo GLP studies Klimisch 2; one human non-GLP study Klimisch 4. No study is selected since a Weight of Evidence determination is made based on human data supported by in vivo data.

Justification for selection of eye irritation endpoint:
Three studies available, one in vitro GLP compliant Klimisch 1; one GLP compliant Klimisch 1 study; one non-GLP study Klimisch 4 where reliability cannot be assigned. Selected study has the highest reliability and is supported by an in vitro study.

Justification for classification or non-classification

The substance does not meet classification criteria under EU Directive 67/548/EEC for dermal irritation.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for dermal irritation.

 

The substance does not meet classification criteria under EU Directive 67/548/EEC for eye irritation.

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for eye irritation.

 

For skin irritation, the weight of evidence indicates that the substance has the potential to be slightly irritating however this is not sufficient for classification purposes. Human experience with the substance indicates the substance does not induce skin irritation. In vivo data indicates both positive and negative irritation however, these studies utilised occlusive dressings (increases percutaneous absorption; potential altering of epidermal lipids, DNA synthesis, epidermal turnover, pH, epidermal morphology) resulting in a predisposition towards positive and irreversible irritation within these studies. In vitro data suggests that the substance is irritating however not corrosive. The overall evidence by expert judgement indicates that the substance may produce transient slight irritation by the dermal route which is not sufficient for classification in accordance with the above criteria.

 

For eye irritation, the weight of evidence clearly indicates the substance is not irritating to the eye.