Reaction mass of (2S,4S,6S,8R)-4,7,8-trimethyltricyclo[5.2.1.02,6]decan-8-ol and (2S,5S,6S,8R)-5,7,8-trimethyltricyclo[5.2.1.02,6]decan-8-ol

Endpoint summary

Description of key information

The skin corrosivity of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 431 using a reconstructed human epidermis method. A mean tissue viability of 104.8% for three minute contact and 91.2% for one hour contact was elicited and was predicted as non-corrosive in the EpiDerm™ skin corrosivity test.
The skin irritation potential of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 439 using a reconstructed human epidermis method. With a mean tissue viability of 9.6 ± 3.1%, the test substance was predicted as irritant to the skin.
The eye irritation potential of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 437 using a Bovine Corneal Opacity & Permeability Assay. The test substance elicited an In Vitro Irritancy Score of 7.0 ± 2.6 and therefore no prediction could be made according to the UN GHS.
The eye irritation potential of the test substance (FRET 08-0338) was assessed according to OECD Test Guideline 405 using an in vivo method. Pannus formation (corneal neovascularisation) is considered to be an irreversible effect; accordingly, FRET 08-0338 was classified as Category 1 in accordance with European Commission regulation 1272/2008.

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:

The study was conducted between 16 December 2014 and 22 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it was conducted according to OECD Test Guideline 439 using a reconstructed human epidermis method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Reconstructed human epidermis skin constructs

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Receipt of tissues
On receipt, the kit contents were checked and the inserts with tissues on agarose were stored at room temperature until use. The kit was used within the expiry date indicated by the supplier (expiry date: 22 December 2014). The maintenance medium was pre-warmed to 37ºC. The tissues were removed from the agar and placed into wells of 12 well plates containing 2 mL pre-warmed maintenance medium per well. The tissues were incubated for a minimum of 24 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.

Type of coverage:

other: Not applicable

Preparation of test site:

other: Not applicable

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

10 µL

Duration of treatment / exposure:

15 minutes

Observation period:

42 hours

Number of animals:

Not applicable

Details on study design:

Reduction of MTT by test substance
It is possible that a test substance may reduce MTT, resulting in the production of a blue colour without any involvement of cellular mitochondrial dehydrogenase. Since the test substance is rinsed off the tissue before the MTT assay, this is usually avoided. However, it is possible that small amounts of test substance may be present after washing or be released through the tissue into the MTT medium. If the mixture with the test substance turns blue/purple after approximately 3 hours incubation at 37 ± 2ºC in 5% CO2 in air, MTT reduction may have occurred.
The MTT reducing capability of the test substance, FRET 08-0338, was investigated by mixing 10 μL of the test substance with 2 mL of 0.3 mg/mL MTT solution in duplicate. A control of 10 μL of purified water, mixed with 2 mL of 0.3 mg/mL MTT solution was also included in duplicate.

Check for colouring potential of test substance
The test substance, FRET 08-0338, was evaluated for its colour or ability to become coloured in contact with water (simulating a tissue humid environment). Evaluation was achieved by mixing 10 μL of the test substance, with 90 μL of purified water in a transparent container.
100 μL of purified water was included as a control. The solution was mixed for 15 minutes on a shaker. At the end of the shaking period the colour of the solution was assessed by eye.

Preparation/application of samples
The test substance, FRET 08-0338, a clear liquid, positive and negative controls were in liquid form and were applied by dispensing a volume of 10 µL over each tissue using a positive displacement pipette.

Test procedure
After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 ± 0.5 minutes with the test substance, negative or positive control at room temperature.
A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30 seconds and then re-spread with a curved flat spatula after 7 minutes application time.
After 15 ± 0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 ± 1 hour at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
After 42 ± 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours ± 5 minutes at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air.
At the end of 3 hours ± 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro-tube.
When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration).
The tissues were extracted by storing at 2-8 ºC, protected from light, for 48 - 70 hours. After formazan extraction, duplicate 200 μL aliquots of the extractant from each tube were pipetted into the wells of flat-bottomed 96-well plates. The extractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

Irritation / corrosion parameter:

other: other: Mean

Value:

9.6

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minutes. Remarks: Predicted as irritant to the skin. (migrated information)

Irritant / corrosive response data:

It was concluded that the test substance, FRET 08-0338, with a mean tissue viability of 9.6 ± 3.1%, was predicted as irritant to the skin.

Check for colouring potential of test substance

The test substance, FRET 08-0338/water solution and water control were colourless after the 15 minute shaking period. The test substance had not shown any potential for colouring water.

 

Assay validity

Negative control

The mean absorbance of the triplicate negative control values was 0.884 which was between the minimum and maximum values of 0.6 and 1.5. The standard deviation (SD) of the % viability was 14.3 which was below the maximum value of 18.

Positive control

The percentage mean viability of the positive control was 14.7 ± 1.1 of the negative control. These were below the maximum acceptance values of 40% viability and SD of 18%.

EpiSkin™ results

The results of the assay are summarised in the table below.

Sample	Tissue viability as percentage of mean OD negative control				Prediction MTT endpoint
	Replicate tissues			Mean±SD
	a	b	c
Negative control	88.	95.4	116.1	100.0±14.3	Not applicable
Positive control	16.0	14.0	14.3	14.7±1.1	Irritant
FRET 08-0338	12.8	6.6	9.4	9.6±3.1	Irritant

 

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It was concluded that the test substance, FRET 08-0338, with a mean tissue viability of 9.6 ± 3.1%, was predicted as irritant to the skin.

Executive summary:

The skin irritation potential of the test substance, FRET 08-0338, was assessed according to OECD Test Guideline 439 using a reconstructed human epidermis method. With a mean tissue viability of 9.6 ± 3.1%, the test substance was predicted as irritant to the skin.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

The study was conducted between 05 June 2015 and 16 June 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 405 using an in vivo method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

The animal for this study was selected from a stock supply of healthy adult rabbits of the New Zealand White strain. The animal weighed 2.71 kg and was approximately 23 weeks of age, prior to treatment (Day 1). She had been acclimatised to the experimental environment for a period of approximately ten weeks prior to the start of the study.
The animal was housed individually in a plastic cage with perforated floors and was offered 150 g of a standard laboratory rabbit diet per day; drinking water was provided ad libitum.
The batch of diet used for the study is analysed for nutrients, possible contaminants and micro-organisms likely to be present in the diet and which, if in excess of specified amounts, might have an undesirable effect on the test system. A dietary supplement of hay was offered during acclimatisation until three days prior to dose instillation, for the remainder of acclimatisation and throughout the study observation period wholemeal bread was offered.
During the acclimatisation and study period the animal was given a small, soft white untreated wood block for environmental enrichment, replaced at appropriate intervals.
Results of routine physical and chemical examination of drinking water, as conducted by the supplier are made available to Huntingdon Life Sciences.
Animal room environmental controls were set to maintain temperature within the range 16 to 20°C, and relative humidity within 40 to 70%. These environmental parameters were recorded and the permanent record archived with other departmental raw data. Lighting was controlled by means of a time switch to give 12 hours of artificial light (06:00 to 18:00 GMT) in each 24 hour period.
The animal was identified by a numbered tag placed through the edge of one ear. This identification was unique within the Department throughout the duration of the study. The cage was identified by a coloured label displaying the study number and animal number.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

The test substance was not removed

Observation period (in vivo):

Seven days after instillation

Number of animals or in vitro replicates:

One

Details on study design:

Treatment procedure
The eyes of the animal were examined prior to instillation of the test substance to ensure that there was no pre-existing corneal damage, iridial inflammation or conjunctival irritation. The animal was gently restrained and the dose was instilled into the right eye by pulling the lower eyelid away from the eye ball to form a cup into which the test substance was dropped. The eyelids were then gently held together for one second before releasing; the left eye remained untreated.
A single animal was treated in advance; the presence of a severe effect in this animal prevented further animals being committed to the study.

Clinical signs
The behaviour of the rabbit was observed immediately following instillation of the test substance to allow assessment of the initial pain response.
The animal was returned to the cage and checked at least twice during the first hour after dosing and at regular intervals throughout the day to ensure no severe injury passed unnoticed. Ocular reactions to treatment were assessed 1, 24, 48 and 72 hours and seven days after treatment, according to the criteria below. Reactions not included overleaf were described in detail.

Ocular responses
The untreated eye was used as a comparison with the treated eye during assessment of ocular lesions.
A pencil beam torch was available for use to facilitate inspection of the eyes.

Termination
Following completion of the observation period the animal was humanely killed by an intravenous injection of sodium pentobarbital.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

1

Reversibility:

not reversible

Remarks on result:

other: Pannus formation (corneal neovascularisation) is considered to be an irreversible effect

Irritation parameter:

iris score

Basis:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

0.3

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

2

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

other: 24, 48 and 72 hours

Score:

0.3

Irritant / corrosive response data:

Ocular responses
A crimson-red conjunctival appearance was evident throughout the study period and very-slight or slight chemosis was apparent during the first 24 hours after instillation, with discharge evident at 1 hour after instillation only. Iritis was evident during the first 24 hours after instillation and also seven days later.
Scattered or diffuse areas of opacity covering up to the entire corneal surface were apparent throughout the first 72 hours after instillation. Seven days after instillation an easily discernible translucent area of opacity covering approximately one quarter of the corneal surface and an area of scattered or diffuse opacity covering approximately half the corneal surface were evident. In addition, two areas of pannus formation (corneal neovascularisation) were apparent; pannus formation is considered to be an irreversible effect and the animal was humanely killed immediately after this observation.
Instillation of the test material gave rise to no initial pain response.

Other effects:

Clinical signs
There was no sign of toxicity or ill health in the study rabbit during the observation period.

Mean values for ocular lesions for Kay and Calandra classification

Mean irritation scores after instillation of FRET 08-0338

Area of eye	1 hour	24 hours	48 hours	72 hours	7 days
Cornea	20	20	20	15	20
Iritis	5	5	0	0	5
Conjunctiva	12	6	4	4	4
Total mean score	37	31	24	19	29

 

Mean values for ocular lesions for EC (Regulation 1272/2008) and GHS classification

24, 48 and 72 hours after instillation of FRET 08-0338

Animal number and sex	Corneal opacity	Iridial lesions	Redness of conjunctiva	Chemosis
188 Female	1.0	0.3	2.0	0.3

 

Grades for ocular irritation responses following instillation of FRET 08-0338

Animal number and sex: 188 F			Pain evaluation response: 0
Region of the eye	Response	Grade of response at time after instillation
		Hours				Day
		1	24	48	72	7
Cornea	Opacity (A)	1	1F+	1F+	1F+	2F+AP
	Area (B)	4	4	6	3	1
	Ulceration	-	-	-	-	-
	Stippling	-	-	-	-	-
Corneal score (A x B x 5)		20	20	20	15	10 + 10
Iris	Value (C)	1	1	0	0	1
Iridial score(C x 5)		5	5	0	0	5
Conjunctiva	Redness (D)	2	2	2	2	2
	Chemosis (E)	2	1	0	0	0
	Discharge (F)	2	0	0	0	0
	Necrosis	-	-	-	-	-
	Ulceration	-	-	-	-	-
Conjunctival score ((D + E + F) x 2)		12	6	4	4	4

F = Female

F+ = Fluorescein positive

A = Additional area of opacity Grade 1 and Area 2 (fluorescein negative)

P = Two areas of pannus formation

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The highest total mean score was 37 occurring at the 1 hour observation; accordingly under the criteria Kay and Calandra (1962) FRET 08-0338 was classified as “moderately irritating” to the eye.
Pannus formation (corneal neovascularisation) is considered to be an irreversible effect; accordingly, FRET 08-0338 was classified as Category 1 in accordance with European Commission regulation 1272/2008.

Executive summary:

The eye irritation potential of the test substance (FRET 08-0338) was assessed according to OECD Test Guideline 405 using an in vivo method. Pannus formation (corneal neovascularisation) is considered to be an irreversible effect; accordingly, FRET 08-0338 was classified as Category 1 in accordance with European Commission regulation 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion/Irritation

Skin corrosion is defined as the production of irreversible damage to the skin, namely visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to 4 hours. Corrosive reactions are typified by ulcers, bleeding, bloody scabs and, by the end of observations at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia and scars.

 

Skin irritation is defined as the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

 

The potential for chemical induced skin corrosion is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. Several factors are considered in determining the corrosion and irritation potential of substances before in vivo testing is undertaken. 

 

To this end, an in vitro study was performed to assess the potential for skin corrosion using the EpiDerm^TMreconstructed human epidermis after treatment periods of 3 minutes and 1 hour

The EpiDerm^TMmodel is a three-dimensional human epidermis skin, consisting of normal, human derived epidermal keratinocytes which have been cultured on 0.6 cm2 inserts to form a multi-layered, highly differentiated model of the human epidermis, with a functional multi-layered stratum corneum.

 

Results showed that the test item did not reduce MTT, and relative mean viabilities of the test item treated tissues were 104.8 % after 3 minutes exposure, and 91.23 % after 1 hour exposure. Therefore the test item is not classified as corrosive.

 

The test item was also assessed for skin irritation using EPISKIN^TMreconstructed human epidermis constructs.The EpiSkin™ Skin Irritation Test was accepted as a replacement to the in vivo Draize Skin Irritation Test, by the European Centre for the Validation of Alternative Methods (ECVAM) in a statement dated 27 April 2007.

 

The test involves the application of the test substance for 15 minutes to the EpiSkin™ three dimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. After 13 days in culture a multi-layered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum is formed.

 

The principle of the assay is that irritant substances are sufficiently cytotoxic to cause cell damage in the cell layers. The cell viability is determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3 (4,5 dimethylthiazol 2 yl) 2, 5 diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify acceptance criteria for both negative and positive controls.

 

The relative mean viability of the test item treated tissues was 9.6 % after a 15-Minute exposure period and was predicted as irritant to the skin.

 

Eye Irritation

Serious eye damage is defined as the production of tissue damage in the eye, or serious physical decay of vision following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application.

 

Eye irritation means the production of changes in the eye following application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application.

The classification system for substances involves a tiered testing and evaluation scheme, and a number of factors are considered in determining eye irritation or serious eye damage.

 

A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. The Bovine Corneal Opacity & Permeability Assay (BCOP) is an in vitro alternative to the in vivo Draize Eye Irritation test. It is an organotypic model that uses isolated bovine corneas from freshly slaughtered cattle as a means of assessing the potential of a test substance to cause serious eye damage.

 

The BCOP test method was evaluated by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), in conjunction with the European Centre for the Validation of Alternative Methods (ECVAM) and the Japanese Center for the Validation of Alternative Methods (JaCVAM), in 2006 and 2010. In the first evaluation (ICCVAM 2006), the BCOP test method was evaluated for its usefulness to identify chemicals (substances and mixtures) inducing serious eye damage. In the second evaluation (ICCVAM 2010), the BCOP test method was evaluated for its usefulness to identify chemicals (substances and mixtures) not classified for eye irritation or serious eye damage.

 

From these evaluations and their peer review it was concluded that the test method can correctly identify chemicals (both substances and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (UN, 2011), and it was therefore endorsed as scientifically valid for both purposes.

 

Two endpoints, namely decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined to generate an In Vitro Irritancy Score (IVIS). The In Vitro Irritancy Score is used to assign an in vitro irritancy hazard classification category for prediction of the ocular irritation potential of a test substance.

 

The results showed that the corneas treated with the test item were opaque post incubation. The In Vitro Irritancy Score was 7.0 ± 2.6 and no prediction of eye irritancy could be made.

 

Further to this, an in vivo eye irritation study was conducted on New Zealand white rabbits. A single animal was treated in advance; the presence of a severe effect in this animal prevented further animals being committed to the study.

A crimson-red conjunctival appearance was evident throughout the study period and very-slight or slight chemosis was apparent during the first 24 hours after instillation, with discharge evident at 1 hour after instillation only. Iritis was evident during the first 24 hours after instillation and also seven days later.

Scattered or diffuse areas of opacity covering up to the entire corneal surface were apparent throughout the first 72 hours after instillation. Seven days after instillation an easily discernible translucent area of opacity covering approximately one quarter of the corneal surface and an area of scattered or diffuse opacity covering approximately half the corneal surface were evident. In addition, two areas of pannus formation (corneal neovascularisation) were apparent; pannus formation is considered to be an irreversible effect.

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted on the target substance, in an appropriate test model and according to internationally recognised guidelines

Justification for selection of eye irritation endpoint:
The study was conducted on the target substance, in an appropriate test model and according to internationally recognised guidelines

Effects on skin irritation/corrosion: irritating

Effects on eye irritation: corrosive

Justification for classification or non-classification

Skin Corrosion/Irritation

Substances are classified as being corrosive and irritant to the skin according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. In vitro skin corrosion and irritation values are expressed as percentage relative mean viabilities.

 

In the skin corrosion test, if the tissue viability from the 3 minute exposure is less than 50% of the negative control value, then the test material is classified as corrosive. If the tissue viability from the 3 minute exposure is greater than or equal to 50% of the negative control value, but the 1 hour value is less than 15% of the negative control value, then the test material is classified as corrosive. If the tissue viability from the 3 minute exposure is greater than or equal to 50% and the 1 hour value is greater than or equal to 15% of the negative control value, then the test material is classified as non-corrosive.

 

The skin corrosion potential of the test substance was assessed in an in vitro skin corrosion test performed on reconstructed human epidermis. The relative mean viability was 104.8 % after the 3 minute exposure and 91.2 % at the 1 hour exposure and therefore the test substance, FRET 08-0338, was not classified for skin corrosion.

 

In the in vitro skin irritation test, if the mean tissue viability was equal to or less than 50% of the negative control value, the sample was classed as an irritant.

 

The skin irritation potential of the test substance was assessed in an in vitro test performed on reconstructed human epidermis. The relative mean viability was 9.6, which indicated that the test substance is a skin irritant.

 

 

Eye Irritation

Substances can be allocated to one of two categories based on irreversible effects on the eye (Category 1) and irritating to the eye (Category 2) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. In vitro eye irritation values were expressed as the In Vitro Irritancy Score (IVIS). An in vitro test was performed using a Bovine Corneal Opacity and Permeability Assay. In this assay, an IVIS ≤3 results in no classification as an eye irritant. An IVIS of > 3 ≤55 indicates that no prediction can be made. An IVIS of > 55 results in a classification as a category 1 eye irritant.

 

The in vitro study performed according to internationally recognized guidelines and conducted to GLP on test item FRET 08-0338 gave an IVIS of 7 and therefore no prediction could be made. 

 

Further to this, an in vivo eye irritation test was performed on New Zealand white rabbits. Irreversible effects on the eye, leading to a classification as a Category 1 irritant are applied if, when applied to the eye of an animal, a substance produces at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or to have not fully reversed within an observation of normally 21 days and / or, at least in two of three tested animals a positive response of a corneal score≥3 and / or iritis > 1.5, calculated as a mean score following grading at 24, 48 and 72 hours.

Following application of the test substance, pannus formation (corneal neovascularisation) was noted. This is considered to be an irreversible effect and therefore the test substance, FRET 08-0338 was classified as a Category 1 for irreversible effects on the eye.