1,5,10-trimethylcyclododeca-1,5,9-triene, epoxidised

Endpoint summary

Description of key information

Skin irritation/corrosion: irritating to the skin (OECD 439, GLP, K, Rel.1)

Eye damage/irritation: Not irritating to the eyes (non OECD, GLP, K, Rel.2)

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 30 September to 05 October 2015

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on June 17, 2015 / Signed on September 24, 2015)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following the REACH bottom-up strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 15-EKIN-039
- Production date: not reported
- Shipping date: 29 September 2015
- Delivery date: 29 September 2015
- Expiry date: 05 October 2015
- Date of initiation of testing: 30 September 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: not reported. At the end of the treatment period, each tissue was rinsed with Dulbeccos Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ to remove any residual test or control items and then incubated in 2mL maintenance medium for 42 h at 37 °C, 5 % CO2 in air.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm (without a reference filter)
- Filter: not applicable
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: no data reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: negative control OD values: 0.704, 0.508 and 0.621 (mean historical OD of the negative control was 0.830; range 0.629 – 1.245.)
The mean OD for the negative control treated tissues narrowly exceeded the lower end of the historical range at 0.611. This was attributed to one of the triplicate negative control tissues producing a low OD of 0.508. However the outlining tissue still produced a clear relative viability of 83.1%.
- Barrier function: IC50 = 2.3 mg/ml ( ≥ 1.5 mg/ml)
- Morphology: Well-differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thinck stratum corneum
- Contamination: absence of bacteria, fungus and mycoplasma
- Reproducibility: For the previous 27 experiments conducted between 14 April 2015 and 21 September 2015 using this test method, the mean OD of the positive control was 0.094; range 0.049 to 0.198 and the mean percentage viability was 11.1; range 4.0 to 23.8 (The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤18%). In this same period the mean OD of the negative control was 0.830; range 0.629 – 1.245 (The assay establishes the acceptance criterion for an acceptable test if the mean OD 562 for the negative control treated tissues was ≥0.6 and ≤1.5).

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): 10 μL (26.3 μL /cm2) of the test item was applied to the epidermis surface.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL

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

Duration of treatment / exposure:

The EpiSkin™ human epidermis skin constructs were treated with the undiluted test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5% CO2 in air for 42 h.

Number of replicates:

Triplicate tissues for test item, negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minute exposure period and 42 h post-exposure incubation period

Value:

36.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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

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.

 

Assessment of Color Interference with the MTT endpoint: The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

 

Table 7.3.1/1: Mean OD₅₆₂Values and Percentage 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	0.704	0.611	0.098	115.2	100*	16.1
	0.508			83.1
	0.621			101.6
Positive Control Item	0.136	0.114	0.032	22.3	18.7	5.3
	0.129			21.1
	0.077			12.6
Test Item	0.306	0.224	0.071	50.1	36.6	11.7
	0.185			30.3
	0.180			29.5

 

SD=Standard deviation; *= The mean viability of the negative control tissues is set at 100%; OD₅₆₂= Optical Density

 

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 18.7% relative to the negative control treated tissues and the standard deviation value of the viability was 5.3. The positive control acceptance criteria were therefore satisfied.

The mean OD₅₆₂ for the negative control treated tissues was 0.611 and the standard deviation value of the viability was 16.1. The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 11.7. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

Under the test conditions, test material is classified as H315 “Causes Skin Irritation” Category 2 according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model. Triplicate tissues were treated with 10 μL of the undiluted test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 h. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. At the end of the formazan extraction period, 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).

This assay was valid with negative and positive controls showing results within the acceptable range.

The test substance with a mean tissue viability of 36.6 ± 11.7 %, was predicted as irritant to the skin. The quality criteria required for acceptance of results in the test were satisfied.

 

Under the test conditions, test material is classified as H315 “Causes Skin Irritation” Category 2 according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

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:

From July 23 to 27, 1979

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)

Principles of method if other than guideline:

Study was performed according to modified Draize procedure (Draize, 1975).

GLP compliance:

no

Remarks:

pre-GLP

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: About 3 months
- Weight at study initiation: Approximately 2 kg
- Housing: Animals were housed in galvanized or stainless steel cages.
- Diet: Diet consisted of a growth and maintenance ration from a commercial producer, ad libitum
- Water, ad libitum
- Acclimation period: 3 days

IN-LIFE DATES: From: July 23, 1979 To: July 27, 1979

Vehicle:

unchanged (no vehicle)

Controls:

other: untreated eye served as control

Amount / concentration applied:

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

Duration of treatment / exposure:

The eyes of all animals remained unwashed for 24 h.

Observation period (in vivo):

24, 48, and 72 h following instillation of the test material

Number of animals or in vitro replicates:

6

Details on study design:

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

SCORING SYSTEM: According to Draize scale.

TOOL USED TO ASSESS SCORE: No data

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

6 animals

Time point:

other: 24, 48 and 72 h

Score:

0.06

Max. score:

4

Reversibility:

fully reversible within: 2 days

Irritation parameter:

iris score

Basis:

mean

Remarks:

6 animals

Time point:

other: 24, 48 and 72 h

Score:

0.06

Max. score:

2

Reversibility:

fully reversible within: 2 days

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

6 animals

Time point:

other: 24, 48 and 72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 3 days

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

6 animals

Time point:

other: 24, 48 and 72 h

Score:

0.44

Max. score:

4

Reversibility:

fully reversible within: 3 days

Irritant / corrosive response data:

Animals showed corneal and iridial reactions which were reversible within 2 days. Conjunctival reactions (redness and chemosis) were reversible within 3 days.

Other effects:

None

Table 7.3.2/1: Mean eye irritation response data of 6 animals at each observation time

Score at time point / Reversibility	Cornea		Iris (/2)	Conjunctivae
	Opacity (/4)	Area (/4)		Redness (/3)	Chemosis (/4)	Discharge (/3)
24 h (Day 1)	0.17	0.17	0.17	0.83	1	0
48 h (Day 2)	0	0	0	0.17	0.33	0
72 h (Day 3)	0	0	0	0	0	0
Mean	0.06	0.06	0.06	0.33	0.44	0
Reversibility	Completely reversible	Completely reversible	Completely reversible	Completely reversible	Completely reversible	-
Average time for reversion	2 days	2 days	2 days	3 days	3 days	-

 

Table 7.3.2/2: Eye irritation response data for each animal at each observation time

 

Score at time point	Cornea		Iris (/2)	Conjunctivae
	Opacity (/4)	Area (/4)		Redness (/3)	Chemosis (/4)	Discharge (/3)
24 h (Day 1)	0 / 0 / 0 / 0 / 0 / 1	0 / 0 / 0 / 0 / 0 / 1	0 / 0 / 0 / 0 / 0 / 1	1 / 1 / 0 / 1 / 1 / 1	1 / 1 / 0 / 1 / 2 / 1	0 / 0 / 0 / 0 / 0 / 0
48 h (Day 2)	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 1	0 / 0 / 0 / 1 / 1 / 0	0 / 0 / 0 / 0 / 0 / 0
72 h (Day 3)	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0	0 / 0 / 0 / 0 / 0 / 0
Average 24h, 48h, and 72h	0 / 0 / 0 / 0 / 0 / 0.33	0 / 0 / 0 / 0 / 0 / 0.33	0 / 0 / 0 / 0 / 0 / 0.33	0.33 / 0.33 / 0 / 0.33 / 0.33 / 0.67	0.33 / 0.33 / 0 / 0.67 / 1.00 / 0.33	0 / 0 / 0 / 0 / 0 / 0
Reversibility	Completely reversible	Completely reversible	Completely reversible	Completely reversible	Completely reversible	-
Average time (unit) for reversion	2 days	2 days	2 days	3 days	3 days	-

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the test material is not classified as irritating to the eyes according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and tot he GHS.

Executive summary:

In an eye irritation study, 0.1 mL of undiluted test material was instilled into one eye of 6 albino rabbits while the contralateral eye remained untreated and served as control. The eyes were not rinsed after the instillation of test material. Animals were observed at 24, 48 and 72 after instillation. The reactions in the conjunctivae (redness, chemosis and discharge), the iris and the cornea (opacity and area involved) were scored according to the Draize Scale.

The calculated mean score for each individual lesion for all the animals within 3 scoring times (24, 48 and 72 h) were: 0.33 for redness, 0.44 for chemosis, 0.06 for iris lesions, 0.06 for corneal opacity and 0.0 for discharge.

The calculated mean score for each individual lesion for each individual animal within 3 scoring times (24, 48 and 72 hrs) were as follows:  0.33 / 0.33 / 0 / 0.67 / 1.00 / for chemosis;  0.33 / 0.33 / 0 / 0.33 / 0.33 / 0.67 for redness; 0/0/0/0 for discharge; 0 / 0 / 0 / 0 / 0 / 0.33 for iris lesions and  0 / 0 / 0 / 0 / 0 / 0.33 for corneal opacity.

Animals showed corneal and iridial reactions which were reversible within 2 days. Conjunctival reactions (redness and chemosis) were reversible within 3 days. 

Under the test conditions, the test material is not classified as irritating to the eyes according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Although some details on experimental conditions were missing, this study is considered as acceptable and satisfies the requirement for eye irritation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (July 2015), was used to evaluate the skin corrosion/irritation potential of the registered substance:

	Element	Information	Conclusion	Comments
Existing data on physico - chemical properties	1a	Is the substance spontaneously flammable in contact with air (pyrophoric) or water at room temperature?	NO
	1b	Is the substance an organic hydroperoxide or an organic peroxide?	NO
	1c	Is the pH of the substance ≤ 2.0 or ≥ 11.5?	NO
	1d	Are there other physical or chemical properties that indicate that the substance is corrosive/irritant?	NO
Existing human data	2	Are there adequate existing human data which provide evidence that the substance is a corrosive or irritant?	NO
Existing animal data from corrosion/irritation studies	3	Are there data from existing studies on corrosion and irritation in laboratory animals, which provide sound conclusive evidence that the substance is a corrosive, irritant or non-irritant?	NO
Existing data from general toxicity studies via the dermal route and from sensitisation studies	4a	Is the substance classified as fatal in contact with skin (LD50 ≤ 50 mg/kg bw, CLP hazard statement H310)	NO
	4b	Has the substance proven to be a corrosive, irritant or non-irritant in a suitable acute dermal toxicity test?	NO	In the acute dermal study, the substance was tested at 50% only. Some skin reactions observed at 24 h were pale red to red, well-defined erythema and barely perceptible edema. Severe desquamation was observed at 7 and 14 days. These observations are not considered sufficient for classification purposes but show that the substance is suspected to be a skin irritant
	4c	Has the substance proven to be a corrosive or an irritant in sensitisation studies or after repeated exposure?	NO
Existing/new (Q)SAR data and read -across	5a	Are there structurally related substances (suitable “read-across” or grouping), which are classified as corrosive to the skin (Skin Corrosive Cat. 1), or do suitable (Q)SAR methods indicate corrosion potential of the substance?	NO
	5b	Are there structurally related substances (suitable “read-across” or grouping), which are classified as irritant to the skin (Skin Irritant Cat. 2), or indicating that the substance is non-irritant, or do suitable (Q)SAR methods indicate irritant or non-irritant potential of the substance?	NO
Existing in vitro data	6a	Has the substance demonstrated corrosive properties in an EU/OECD adopted in vitro test? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met	NO
	6b	Has the substance demonstrated irritant or non-irritant properties in an EU/OECD adopted in vitro test? Data from in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by EU and/or OECD may also be used if the provisions defined in Annex XI are met.	NO	(at the initiation of the dossier, no test was available)
	6c	Are there data from a non-validated suitable in vitro test(s), which provide sound conclusive evidence that the substance is corrosive/ irritant?	NO
Weight-of- Evidence analysis	7	The “elements” described above may be arranged as appropriate. Taking all available existing and relevant data mentioned above (Elements 1-6) into account, is there sufficient information to make a decision on whether classification/labelling is necessary, and –if so –how to classify and label?	NO
New in vitro test for irritation	8	Does the substance demonstrate irritating or non-irritating properties in (an) EU/OECD adopted in vitro test(s) for skin irritation?	YES	=> an Episkin test for irritation was initiated (Bottom-up strategy - substance expected to be non corrosive). Together with the absence of eye irritation in vivo, the conclusion of this Episkin test was considered sufficient to conclude on C&L and a skin corrosion test was not performed.
New in vitro test for corrosivity	9	Does the substance demonstrate corrosive properties in (an) EU/OECD adopted in vitro test(s) for skin corrosion?	NO
New in vivo test for corrosion/irritation	10	To be used only as a last resort	NO	In vivo testing should not be conducted in this case since the substance falls under the scope of the specific in vitro tests performed, and there are no substance-specific limitations on use of those tests. An adaptation according to Annex XI to the REACH Regulation is included in this dossier.

 

The purpose of the newly performed in vitro test (Envigo, 2016, rel.1) was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. This test was performed in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the test item-treated tissues was 36.6 ± 11.7 %, after the 15‑minute exposure period. With a tissue viability < 50%, the registered substance was considered to be irritating to skin.

Together with the absence of eye irritation in vivo, the conclusion of this Episkin test was considered sufficient to conclude on C&L and a skin corrosion test was not performed.

Eye irritation:

In an eye irritation study (Consumer product testing, 1979, non OECD, GLP, Rel. 2, K), 0.1 mL of undiluted test material was instilled into one eye of 6 albino rabbits while the contralateral eye remained untreated and served as control. The eyes were not rinsed after the instillation of test material. Animals were observed at 24, 48 and 72 after instillation. The reactions in the conjunctivae (redness, chemosis and discharge), the iris and the cornea (opacity and area involved) were scored according to the Draize Scale.

The calculated mean score for each individual lesion for all theanimals within 3 scoring times (24, 48 and 72 h) were: 0.33 for redness, 0.44 for chemosis, 0.06 for iris lesions, 0.06 for corneal opacity and 0.0 for discharge.

The calculated mean score for each individual lesion for each individual animal within 3 scoring times (24, 48 and 72 hrs) were as follows:  0.33 / 0.33 / 0 / 0.67 / 1.00 /for chemosis;  0.33 / 0.33 / 0 / 0.33 / 0.33 / 0.67 for redness; 0/0/0/0 for discharge;0 / 0 / 0 / 0 / 0 / 0.33 for iris lesions and  0 / 0 / 0 / 0 / 0 / 0.33 for corneal opacity.

Animals showed corneal and iridial reactions which were reversible within 2 days. Conjunctival reactions (redness and chemosis) were reversible within 3 days. 

The test material does not require classification for eye irritation.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self classification:

Skin irritation:

Based on the available information, the substance should be classified as Skin irr. Category 2 (H315: Causes skin irritation) according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

Eye irritation:

Based on the available information on the substance, no additional self-classification is proposed regarding eye irritation according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

Respiratory irritation:

No data was available.