2-benzyl-4,4,6-trimethyl-1,3-dioxane

Endpoint summary

Description of key information

Skin corrosion: No corrosive effects are expected based on absence of such effects in an acute dermal toxicity study (tested up to 5000 mg/kg bw) and in a LLNA tested up to 100%.

Skin irritation (OECD TG 439): irritating

Eye irritation (OECD TG 438): not irritating

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:

24-30 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

In accordance with GLP conditions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2009

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN TM
- Tissue batch number(s): 16-EKIN-021
- Delivery date: 24 May 2016
- Date of initiation of testing: 24 May 2016

PRE-TEST PROCEDURE
- Pre-incubation: On day of receipt the tissues were transferred into a 12-wells plate filled with maintenance medium. A different 12-wells plate was used for the test and each control item were. The tissue were incubated at 37°C, 5% CO2 in air overnight.
- Test item colour interference: To assess colour interference, 10 μL of Peomosa (mono-constituent) was added to 90 μL sterile water. The mixture was mixed for approximately 15 minutes. At the end of the shaking period a colour check was performed.
- Test item MTT reduction: To assess the ability of the test item to reduce MTT, 10 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, 5% CO2 in air. Untreateed MTT solution was used as a control. At the end of the incubation period a colour check was performed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure/ post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: not specified
- Wavelength: 562 nm (without reference fildeter)

NUMBER OF REPLICATE TISSUES: 3

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubat
ion is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of
post incubation is > 50% of the mean viability of the negative controls.

ACCEPTANCE CRITERIA
- Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated
tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
- Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
- Test Item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

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

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

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

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Relative mean tissue viability compared to the negative control tissues (100%)

Value:

26.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4.9

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: No colour changes observed
- Colour interference with MTT: Not colour changes observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: No data

ACCEPTANCE OF RESULTS:
-The relative mean tissue viability for the positive control treated tissues was 4.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.9%. The positive control acceptance criteria were therefore satisfied.
-The mean OD562 for the negative control treated tissues was 1.293 and the standard deviation value of the viability was 3.5%. 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 1.0%. The test item acceptance criterion was therefore satisfied.

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	1.265 / 1.345 /1.269	1.293	0.045	97.8 / 104.0 / 98.1	100∗	3.5
Positive Control Item	0.076 / 0.058 / 0.055	0.063	0.011	5.9 / 4.5 /4.3	4.9	0.9
Test Item	0.365 / 0.333 / 0.355	0.348	0.013	27.5 / 25.8 / 27.5	26.9	1.0

OD = Optical Density

SD = Standard deviation

∗ = The mean viability of the negative control tissues is set at 100%

⊕ = The control groups were shared with Envigo - Shardlow study number TW67BS, MS48XJ, FC59GT, LL32QY, RS46VL, JD61DM and NL15CD.

Interpretation of results:

other: Category 2 (skin irritant)

Remarks:

based on CLP criteria (EC 1272/2008 and its amendments)

Conclusions:

Under the conditions of this test, the relative mean tissue viability for the test item determined to be 26.9%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, it can be concluded that Reseda body is an irritant to skin (Category 2 according to the CLP Regulation).

Executive summary:

The possible skin irritation potential of Reseda body 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 10 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 4.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item determined to be 26.9%. This value is below the threshold for irritancy of ≤50%.

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:

22-03-2016 to 04-04-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

In accordance with GLP

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)

Remarks:

Triskelion, 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:

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:

other: Positive controls: Benzalkonium Chloride. Negative control: Physiological saline.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

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. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes

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.

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 value

Irritation parameter:

cornea opacity 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 value

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

Other effects / acceptance of results:

Slit-lamp examination: The test substance did not cause any corneal effects, other than very slight swelling (mean of 1%). 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 moderate or 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 did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas).

Interpretation of results:

other: not eye irritating

Remarks:

based on EU CLP criteria (1272/2008 and its amendments)

Conclusions:

Under the test conditions (OECD 438 and GLP) the test substance is not considered to be an eye irritant.

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 did not cause any corneal effects, other than very slight swelling (mean of 1%). 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 moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas). Based on these results, the test substance is considered to be not eye irritating.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin corrosion

Based on the results from an acute dermal toxicity study (tested up to 5000 mg/kg bw) and a LLNA tested up to 100%, which both didn't show any corrosive effects, the substance is not considered corrosive.

Skin irritation (in vitro)

The possible skin irritation potential of Reseda body 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 10 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 4.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item determined to be 26.9%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, it can be concluded that Reseda body is an irritant to skin.

Eye irritation (in vitro)

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 did not cause any corneal effects, other than very slight swelling (mean of 1%). 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 moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas). Based on these results, the test substance is considered to be not eye irritating.

Justification for classification or non-classification

Skin: Based on the positive results found in the EpiSkin test and the supporting information, Reseda body is considered to cause skin irritation. In accordance with the criteria outlined in EU CLP (1272/2008/EC and its amendments), this results in a Category 2 classification for this endpoint (H315 / Causes skin irritation).

Eye:Based on the negative results found in the in vivo eye irritation test, Reseda body does not need to be classified as an eye irritant in accordance with the criteria outlined in EU CLP (1272/2008/EC and its amendments).