[3R-(3α,3aβ,6α,7β,8aα)]-octahydro-6-methoxy-3,6,8,8-tetramethyl-1H-3a,7-methanoazulene

Endpoint summary

Description of key information

Skin irritation (OECD TG 404): Not irritating.

Eye irritation OECD TG 438): Not eye irritating

Respiratory irritation: Not respiratory irritating in view of the absence of irritation for skin and eye.

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:

2015-09-02 until 2015-09-07

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)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

Preliminary tests:
- Some chemicals are known to change into a coloured substance in aqueous conditions and consequently stain tissues during the exposure. To determine this possibility, 30 μL of the test substance was incubated in 300 μL MilliQ water for 60 min at ca. 37 ºC and 5% CO2 and a colour change was assessed visually. Some chemicals are known to non-specifically reduce MTT, resulting in a blue/purple precipitate and/or blue/purple staining of a MTT solution. To test the MTT reducing capacity of the test substance, 30 μL of the test substance was incubated in 1 mL of a MTT solution (1 mg/mL) for 185 min at ca. 37ºC and 5% CO2 and the formation of a blue/purple formazan product was assessed visually. To test if the test substance had the potential to damage a nylon mesh, a mesh compatibility test was performed in which 30 μL of the test substance was applied to a nylon mesh. After 60 min incubation at ambient temperature, the possible interaction with the mesh was visually checked using a microscope.

Exposure to study substances:
- The skin models were topically exposed to 30 μL of the test substance, negative or positive control, at the end of the acclimatization period. Immediately after application a nylon mesh was placed on the skin model surface to facilitate an equal distribution of the study substances. Exposure was initiated at ambient temperature. After dosing the last skin model, all skin models were transferred to a humidified incubator (ca. 37ºC and 5% CO2). After 36 min, the plates were removed from the incubator and kept at ambient temperature until the exposure period of 60 min was completed. Subsequently, the skin models were removed from the well, washed using an excess of PBS to remove the study substances and mesh. The skin models were transferred to a clean 6-well plate containing fresh NMM (900 μL/well) and incubated in a humidified incubator (ca. 37ºC and ca. 5% CO2). Medium was refreshed after 18 h post-exposure. Following an additional 24 h incubation period (i.e. the total post-exposure period was 42 h), viability was determined using the MTT test.

MTT test:
- The MTT solution of 1 mg/mL was freshly prepared by diluting MTT concentrate five times in MTT diluent. The inserts were transferred to a 24-well plate containing 300 μL of MTT solution per well. After 178 min incubation in a humidified incubator at ca. 37 ºC and 5% CO2, the skin models were rinsed three times with PBS. The formazan product was extracted from the skin model using 2 mL MTT extractant (provided with the MTT-100 assay kit). Extraction was performed at 2-10 ºC for three days. Following extraction, the optical density was measured in triplicate in 200 μL sub fractions per well in a 96-well plate using a spectrophotometer set at 570 nm. MTT extractant was used as blank. The mean optical density (OD) was calculated and expressed as the percentage viability compared to the negative control (mean tissue viability).

Interpretation of results:
The test was considered valid if
- the OD of the negative control was ≥ 0.8 and ≤ 2.8
- the OD of the blank was < 0.1
- skin models treated with the positive control showed mean tissue viability ≤ 20% compared to the negative control
The test was considered invalid if the test did not meet these acceptance criteria.

- The test group was considered valid if the SD calculated from individual tissue viability percentages of the three replicates was ≤ 18%.
- Test substances showing tissue viabilities in a range of 30-70% may show standard deviation (SD) > 18%. If this was typical for the test substance, and consistent in a repeat experiment, the results were accepted, although the acceptance criterion
was not met.
- The test group was considered invalid (inconclusive) if the SD calculated from individual tissue viability percentages of the three replicates was >18% and if the test was not repeated.
The in vitro irritation potential of the test substance was determined from the relative mean tissue viabilities compared to the negative control tissues, using the following prediction model:

Mean tissue viability (% of negative control) Prediction
Mean tissue viability ≤ 50 % Irritant
Mean tissue viability > 50 % Non-irritant (No Category)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 µL

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

Viability of the epidermal cells was assessed using the MTT test after 42h of culture.

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Value:

94

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Preliminary tests:
- At the end of the incubation period of the test substance in MilliQ, the solution did not significantly changed colour to blue/purple, indicating that there would be no colour interference in the test. Therefore, no additional controls were required in the in vitro skin irritation test.
- At the end of the incubation period of the test substance with a MTT solution, the MTT solution had neither turned blue/purple nor showed a blue/purple precipitate, indicating that the test substance did not have the potential to reduce MTT. Therefore, no additional controls were required in the in vitro skin irritation test. During the mesh compatibility test it was observed that the test substances did not damage the nylon mesh and therefore the nylon mesh was used in the in vitro skin irritation test to facilitate equal distribution of the test substance.

In vitro skin irritation test:
- The OD of the blank, the negative control (PBS) and the positive control (5% SDS) demonstrated the expected response. The SD calculated from individual tissue viability percentages of the three replicates was <18%. All acceptance criteria were met and therefore the study was considered valid.

Interpretation of results:

other: Not a skin irritant

Remarks:

in accordance with EU CLP 1272/2008 and its amendments

Conclusions:

Under the test conditions (OECD 439 and GLP) the test substance is not a skin irritant in accordance with EU CLP and GHS.

Executive summary:

In accordance to OECD guideline 439 and GLP the test substance was examined for its in vitro skin irritation potential using EpiDerm™ reconstructed skin membranes. In the in vitro skin irritation test, the skin membranes were topically exposed to the undiluted test substance for 60 min. The test was performed in triplicate. Viability of the epidermal cells was assessed using the MTT test after 42 h of culture. Negative and positive controls were run in parallel. All acceptance criteria were met and therefore the study was considered valid. After exposure to the test substance the mean tissue viability was 94 ± 17 % compared to the concurrent negative control group. Based on the results obtained in the present study the test substance is not considered to be a skin irritant, not for EU CLP and not for GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

2015-09-01 until 2015-09-29

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 Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Species:

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

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: approximately 1.5 - 2.5 kg
- 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.
- The preparation and validation of the eyes prior to the ICE-test were all according to OECD guideline 438.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 µL

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

0, 30, 75, 120, 180, and 240 minutes

Number of animals or in vitro replicates:

3 eyes

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The eyes were rinsed with 20 mL saline
- Time after start of exposure: 10 seconds

SCORING SYSTEM: According to OECD 438 guideline.

TOOL USED TO ASSESS SCORE: All examinations were carried out with the hand-slit lamp microscope. Fluorescein retention was only scored at approximately 30 minutes after treatment. 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.

CONTROLS: A negative control (30 µL physiological saline) and 3 positive controls (30 µL Benzalkonium Chloride 5%) were included.

Irritation parameter:

percent corneal swelling

Run / experiment:

slit-lamp examination

Value:

1

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:

0.8

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

Irritant / corrosive response data:

- Slit-lamp examination:
The test substance caused very slight corneal swelling (1%), very slight or slight opacity (mean score of 0.8) and no fluorescein retention (mean score of 0.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
- Microscopic examination:
Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion (two corneas) and slight vacuolation (one cornea) of the epithelium. In addition, the epithelium was partly detached form the basement membrane in the same cornea. In view of the slit-lamp observations of this cornea, the toxicological significance of these findings was considered dubious and not taken into account for the classification. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities, apart from very slight vacuolation (top region) of the epithelium. The positive control BAC 5% caused moderate or severe erosion, slight vacuolation (one cornea; low region) of the epithelium, and endothelial necrosis.

Interpretation of results:

other: Not eye irritating

Remarks:

EU CLP 1272/2008 and its amendments

Conclusions:

Under the test conditions (OECD 438 and GLP) the test substance is not an eye irritant using the EU CLP and GHS criteria.

Executive summary:

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. 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. The test substance caused very slight corneal swelling (1%), very slight or slight opacity (mean score of 0.8) and no fluorescein retention (mean score of 0.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. 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 treated with the test substance revealed very slight or slight erosion (two corneas) and slight vacuolation (one cornea) of the epithelium. In addition, the epithelium was partly detached form the basement membrane in this cornea. In view of the slit-lamp observations of this cornea, the toxicological significance of these findings was considered dubious and not taken into account for the classification. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities, apart from very slight vacuolation (top region) of the epithelium. The positive control BAC 5% caused moderate or severe erosion, slight vacuolation (one cornea; low region) of the epithelium, and endothelial necrosis. Based on these results the test substance is not considered to be an eye irritant in accordance with EU CLP and GHS criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation:

In accordance to OECD guideline 439 and GLP the test substance was examined for its in vitro skin irritation potential using EpiDerm™ reconstructed skin membranes. In the in vitro skin irritation test, the skin membranes were topically exposed to the undiluted test substance for 60 min. The test was performed in triplicate. Viability of the epidermal cells was assessed using the MTT test after 42 h of culture. Negative and positive controls were run in parallel. All acceptance criteria were met and therefore the study was considered valid. After exposure to the test substance the mean tissue viability was 94 ± 17 % compared to the concurrent negative control group. Based on the results obtained in the present study the test substance is not considered to be a skin irritant in accordance with EU CLP and GHS criteria.

Eye irritation:

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. 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. The test substance caused very slight corneal swelling (1%), very slight or slight opacity (mean score of 0.8) and no fluorescein retention (mean score of 0.0). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. 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 treated with the test substance revealed very slight or slight erosion (two corneas) and slight vacuolation (one cornea) of the epithelium. In addition, the epithelium was partly detached form the basement membrane in this cornea. In view of the slit-lamp observations of this cornea, the toxicological significance of these findings was considered dubious and not taken into account for the classification. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities, apart from very slight vacuolation (top region) of the epithelium. The positive control BAC 5% caused moderate or severe erosion, slight vacuolation (one cornea; low region) of the epithelium, and endothelial necrosis. Based on these results the test substance is not considered to be an eye irritant.

Respiratory irritation:

For assessing respiratory irritation human data are needed because no suitable in vitro or in vivo tests are available that can identify respiratory irritation (ECHA guidance R.7.2.1.1, 2014). There are no human data such as indicated in R7a, 7.2.3.2 and 7.2.4.2 of the ECHA guidance (2014) that indicate respiratory reactions of the substance e.g. from consumer experience or occupational exposure. In view of the absence of skin and eye irritation (ECHA guidance (7.1.2.1, last paragraph) respiratory irritation is not anticipated.

Justification for classification or non-classification

Based on the absence of skin and eye irritation the substance does not have to be classified for skin, eye and respiratory irritation in accordance with CLP, Regulation (EC) No.1272/2008 and its amendments as well as in accordance with GHS criteria