Bicyclo[3.1.1]hept-2-ene, 2,6,6-trimethyl-, phosphosulfurized

Endpoint summary

Description of key information

An acute toxicity study by dermal route was performed on the registered substance, which showed no skin corrosion and a slight irritation after a 24-hour exposure. Therefore, it was not considered justified to perform an in vitro skin corrosion study.
The skin irritation study was conducted on the registered substance according to OECD Testing Guideline 439 and gave a relative mean tissue viability calculated as a percentage of the negative control of 110.9 % after the 15-minute exposure followed by the 42-hour post-exposure. The registered substance is not classified as a skin irritant according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.
The eye irritation study was conducted on the registered substance according to a method similar to OECD Testing Guideline 405 and induced a slight redness in all animals, fully reversible within 24 hours for 4/6 animals. The registered substance did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

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:

From 17 February 2016 to 22 February 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: E00350-290
- Expiration date of the lot/batch: 23 June 2019
- Purity test date: 58 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Test system:

human skin model

Remarks:

EPISKIN reconstructed human epidermis

Source species:

human

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKINTM Reconstructed Human Epidermis Model Kit
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 16 February 2016
EpiSkinTM Tissues (0.38cm²) lot number: 16-EKIN-007
Maintenance Medium lot number: 16-MAIN3-009
Assay Medium lot number: 16-ESSC-007

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL (26.3 µL/cm²)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL Dulbecco’s Phosphate Buffered Saline with Ca++ and Mg++

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL Sodium Dodecyl Sulphate
- Concentration (if solution): 5 % w/v aqueous solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean

Value:

110.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

113

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

106.4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

113.3

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was colourless, it was therefore unnecessary to run color correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES
- Acceptance criteria met for positive control: YES
- Acceptance criteria met for variability between replicate measurements: YES

Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD562 of tissues	Mean OD562 of triplicate tissues	±SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	±SD of relative mean viability
Negative Control Item	0.865	0.845	0.024	102.3	100*	2.8
	0.819			96.9
	0.852			100.8
Positive Control Item	0.063	0.071	0.012	7.5	8.4	1.3
	0.065			7.7
	0.084			9.9
Test Item	0.955	0.937	0.033	113.0	110.9	3.9
	0.899			106.4
	0.957			113.3

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

Interpretation of results:

GHS criteria not met

Conclusions:

The relative mean tissue viability calculated as a percentage of the negative control was 110.9 % after the 15-minute exposure followed by the 42-hour post exposure. The test substance did not meet the criteria for classification according to Regulation (EC) No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The in vitro skin irritation of the test substance was determined in accordance with the OECD Testing Guideline 439. The purpose of this test was to evaluate the skin irritation potential of the registered substance using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the registered substance treated tissues relative to the negative controls.

Triplicate tissues were treated with the registered substance for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean tissue viability calculated as a percentage of the negative control was 110.9 % after the 15-minute exposure followed by the 42-hour post-exposure. The test substance did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1970

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

missing data on animals

Principles of method if other than guideline:

Study was conducted using a method equivalent to OECD Testing Guideline 405 and meets acceptable scientific standards. 0.1mL of the registered substance was instilled in the left eye of six New Zealand White, which were therefore observed for seven days, as all observable adverse effects had disappeared by this time. Observations were recorded and scored according to Draize, which is the scoring system used by EU and OECD methods. As a result, it is considered that the study is providing a sufficient amount of data in order to conclude regarding the eye irritation of the registered substance.

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 2.6 - 3.1 kg

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

Duration of treatment / exposure:

Treated eye held closed for one second following application

Observation period (in vivo):

7 days (it was not considered relevant to pursue the observation as all effects had reversed).

Number of animals or in vitro replicates:

6

Details on study design:

SCORING SYSTEM: According to J.H. Draize

TOOL USED TO ASSESS SCORE: 2.0 % sodium fluorescein stain

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

cornea opacity score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

iris score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48h

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 48h

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24h

Remarks on result:

other: Max score of 1 reported at 1 and 4 hours observation

Irritation parameter:

conjunctivae score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24h

Remarks on result:

other: Max score of 1 reported at 1 and 4 hours observation

Irritation parameter:

conjunctivae score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24h

Remarks on result:

other: Max score of 1 reported at 1 and 4 hours observation

Irritation parameter:

conjunctivae score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

1

Reversibility:

fully reversible within: 24h

Remarks on result:

other: Max score of 1 reported at 1 and 4 hours observation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #4

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #5

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritation parameter:

chemosis score

Basis:

animal #6

Time point:

24/48/72 h

Score:

0

Max. score:

0

Irritant / corrosive response data:

Slight redness observed at 1 hour and 4 hours in all animals and at 24 hours in two animals. Slight discharge appeared in all animals at one hour only. All effects were fully reversible in all animals and fluorescein staining at seven days confirmed the absence of corneal damage in all eyes.

Other effects:

Blinking in all animals after treatment.

See attached background information.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance induced a slight redness in all animals, fully reversible within 24 hours for 4/6 animals and 48 hours for 6/6 animals. The test substance did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The test substance was analysed for eye irritation on New Zealand white rabbit eyes using a method similar to OECD Guideline 405. The left eye of the animals was exposed to 0.1 mL of the test substance whilst the right eye was left as a control. Fluorecein staining at seven days showed no corneal damage in any of the test subjects. Slight redness was observed in all animals after exposure but did not last more than a day. All animals also showed slight discharge at one hour only. All effects were reversible within 24 hours for 4/6 animals and 48 hours for the remaining animals. The test substance did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The skin irritation potential of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 439. The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The Relative mean tissue viability calculated as a percentage of the negative control was 110.9 % after the 15-minute exposure followed by the 42-hour post-exposure.

The eye irritation potential of the registered substance was determined in accordance with a method similar to OECD Guideline for Testing of Chemicals 405 with deviation. The left eye of six New Zealand White rabbits were exposed to 0.1 mL of the test substance whilst the right eye was left untreated and used as a control. Fluorecein staining at seven days showed no corneal damage in any of the test subjects. A slight redness at 1 hour and 4 hours in all animals and at 24 hours in two animals. Slight discharge appeared in all animals at one hour only. The observed effects were fully reversible.

Justification for selection of skin irritation / corrosion endpoint:
Study was conducted on the registered substance according to OECD Testing Guideline 439. According to the Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 4.0), reconstructed human epidermis (RHE) tests are considered scientifically valid for the prediction of irritant (Category 2) and non-irritant (no category) substances in accordance with the rules laid down in Annex XI, and therefore additional in vivo testing is not considered as scientifically justified.

Justification for selection of eye irritation endpoint:
Study was conducted using a method equivalent to OECD Testing Guideline 405 and meets acceptable scientific standards and the study was conducted on the registered substance.

Justification for classification or non-classification

The skin irritation study was conducted on the registered substance according to OECD Testing Guideline 439 and gave a relative mean tissue viability calculated as a percentage of the negative control of 110.9 % after the 15-minute exposure followed by the 42-hour post-exposure. The test substance is not classified as a skin irritant according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

The eye irritation study was conducted on the registered substance according to a method similar to OECD Testing Guideline 405 and induced a slight redness in all animals, fully reversible within 24 hours for

4/6 animals. The test item did not meet the criteria for classification according to Regulation (EC) No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.