Reaction mass of 4-methyl-1-oxaspiro[5.5]undec-3-ene and 4-methyl-1-oxaspiro[5.5]undec-4-ene and 4-methylidene-1-oxaspiro[5.5]undecane

Endpoint summary

Description of key information

Skin corrosion: The substance is not corrosive based on absence of highly reactive groups such as acids or base and in absence of eye irritation.

Skin irritation (OECD TG 439): Irritating Cat.2

Eye irritation (OECD TG 438): Not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2017 - 16 October 2017

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)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: Not applicable

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- EPISKIN Small Model (TM) (EPISKIN-SM (TM), 0.38 cm^2, Lot no.: 17-EKIN-041); a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen and cultured for 13 days.
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 2.5 hours at 37°C.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.6 - 37.1 °C
- Humidity (%): 65 - 85

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed once with phosphate buffered saline
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, 25 μL sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
- Cell viability: The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw): ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u +MTT).
The % viability for each sample and the positive control is calculated as follows: %Viability = (ODc/mean ODlt_u+MTT) * 100
- Skin irritation is expressed as the remaining cell viability after exposure to the test item.

ACCEPTABILITY CRITERIA:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
b) The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM:
25 μL directly on top of the tissue

NEGATIVE CONTOL:
- Amount applied: 25 μL Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 μL
- Concentration: 5% (aq) Sodium dodecyl sulphate
- Re-spread after 7 minutes contact time

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours and 3 hours with MTT

Number of replicates:

3 for the test item, the negative and the positive control, each.

Irritation / corrosion parameter:

% tissue viability

Value:

12

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Cell viability: 10%

Other effects / acceptance of results:

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the three tissues of the negative control were within the laboratory historical control data range (i.e., a mean of 1.115 ± 0.051) and the SD of the % viability was <18% (i.e., 4.5%)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e., 10%) and the SD of the % viability was <18% (i.e., 0.8%).
- Acceptance criteria met for variability between replicate measurements: yes, the SD calculated from individual % tissue viabilities of the three identically treated replicates was <18% (i.e., 5%).

- Since the mean relative tissue viability for the substance was below 50%, it is considered to be irritant.

Table Individual OD measurements (570 nm)

	A (OD570)	B (OD570)	C (OD570)
Negative control OD570measurement 1 OD570measurement 2	1.2414 1.1881	1.2934 0.9755	1.2003 1.0412
Test item OD570measurement 1 OD570measurement 2	0.2161 0.2166	0.2142 0.1402	0.1344 0.1054
Positive control OD570measurement 1 OD570measurement 2	0.1637 0.1397	0.1728 0.1625	0.1501 0.1536

OD = Optical density

Triplicate exposures are indicated by A, B and C.

Interpretation of results:

other: Skin irritant Cat. 2

Remarks:

according to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

An in vitro skin irritation test with the substance was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the substance is irritating in the in vitro skin irritation test.

Executive summary:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. All validity criteria were met and the study was considered to be valid. The positive control had a mean cell viability of 10% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 12%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be irritant to the skin.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

07 Nov 2017 to 16 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3700 AV, Zeist

Species:

other: Eyes of male or female chickens (ROSS, spring chickens)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Characteristics of donor animals: Approximately 7 weeks old, male or female chickens, body weight range approximately 1.5-2.5 kg, were used as eye donors.
- Storage, temperature and transport conditions of ocular tissue: Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 μL neat substance

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

Test group and positive control: triplicates
Negative control: Singlo

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short.
The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C (water pump set at 36.4 °C). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline. After rinsing, each eye in the holder was returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp microscope examination
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope examination
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: set at 0.095 mm
- Others: After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

SCORING SYSTEM:
Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV).
- Mean corneal swelling (%): According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean maximum opacity score: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean fluorescein retention score at 30 minutes post-treatment: According to OECD 438 guideline.

DECISION CRITERIA: According to OECD 438 guideline

Irritation parameter:

percent corneal swelling

Run / experiment:

slit-lamp examination

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

cornea opacity score

Run / experiment:

slit-lamp examination

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

fluorescein retention score

Run / experiment:

slit-lamp examination

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Other effects / acceptance of results:

Slit-lamp examination: The test substance only caused very slight corneal swelling (mean score 2%) and very slight corneal opacity (mean score 0.5). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the test were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Interpretation of results:

other: Not an eye irritant

Remarks:

according to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

Under the test conditions (OECD 438 and GLP) the test substance is not classified as eye-irritant.

Executive summary:

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, chicken eyes were exposed to 30 µL test substance for 10 seconds. In addition, the test included a negative control eye (30 µL saline) and a positive control eye (30 µL 5% Benzalkonium Chloride (BAC)). The isolated chicken eyes were exposed to a single application of 30 μl of the test substance for 10 seconds. After the exposure, the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. Mean fluorescein retention score was determined at 30 minutes post-treatment. The test substance caused very slight corneal swelling (mean score 2%) and very slight corneal opacity (mean score 0.5). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea did not reveal any abnormalities.

The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas generally revealed severe erosion and very slight or slight vacuolation of the epithelium and the epithelial layer (partly) detached from the basal layer. Based on these results, the substance does not need to be classified as irritating to the eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin corrosion:The substance is not considered to be corrosive because it does not contain reactive groups (e.g. acid or base) that would indicate corrosion. The substance is a hydrocarbon with an ether bond as a functional group, which are known to be non-corrosive. In addition, the substance is not an eye irritant further indicating that corrosion is unlikely.

In vitro skin irritation:

The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 10% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 12%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be irritant to the skin. All validity criteria were met and the study was considered to be valid.

In vitro eye irritation:

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, chicken eyes were exposed to 30 µL test substance for 10 seconds. In addition, the test included a negative control eye (30 µL saline) and a positive control eye (30 µL 5% Benzalkonium Chloride (BAC)).The isolated chicken eyes were exposed to a single application of 30 μl of the test substance for 10 seconds.After the exposure, the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. Mean fluorescein retention score was determined at 30 minutes post-treatment. The test substance caused very slight corneal swelling (mean score 2%) and very slight corneal opacity (mean score 0.5). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea did not reveal any abnormalities.

The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas generally revealed severe erosion and very slight or slight vacuolation of the epithelium and the epithelial layer (partly) detached from the basal layer. Based on these results, the substance does not need to be classified as irritating to the eyes.

Justification for classification or non-classification

Based on the information above the substance is as a skin irritatn Cat. 2 and shall be labelled with H315: Causes skin irritation, according to EU CLP (EC 1272 /2008 and its amendments), but not for eye irritation.