Dodecane-1,12-diol

Endpoint summary

Description of key information

Skin irritation/corrosion

An in vitro study for predicting non-specific irritating properties of the test item was conducted according to OECD TG 439. The undiluted test item (30 mg per insert) was applied topically to a reconstructed human epidermis model (RhE; epiCS). The cell viability was determined with 95.28 % for the test item as measured by MTT conversion. It is therefore concluded that the test item is not irritating to skin.

Eye Irritation

In an in vivo eye irritation study with the test substance performed according to OECD 405 on six rabbits (3/sex), the test substance induced no irritation when applied into the rabbit eye mucosa. No acute toxicological signs were observed. Because no further information on the test substance identity is given in the study report, rather than "Dodecandiol", an in vitro repetion with a well characterized test item was performed.

The EpiOcularTM Eye Irritation Test (EIT) conducted according to OECD TG 492 confirmed the in vivo result for the well characterized tes item. The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hours post-treatment incubation period. Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %.

The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model. The final mean percent tissue viability recorded for the test item is 62 % (rounded).

According to the results of this study the test item was identified as not requiring classification for eye irritation according to UN GHS (No Category).

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

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:

2015

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

commercially available test system

Vehicle:

other: ethyl acetate

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Tissue batch number(s): Cat.-No.CS-1001
- Date of initiation of testing: 01 August 2019
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT
- Temperature of post-treatment incubation (of 42 h): 37 ± 2 °C
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
the optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.
PREDICTION MODEL / DECISION CRITERIA:
- The mean optical density (OD) value obtained with the test item was used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- A category of UN GHS (Category 2 or Category 1) is predicted if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

The irritating potential of the test item is assessed by determination of its cytotoxic effect on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay reflecting the cell viability after exposure to the topically applied test item.
All tests were performed in triplets for each time point. The test item was applied at a 100 % concentration, i.e. 30 mg per insert for 20 min. Cell viability was measured by the amount of MTT reduction (calculated on the basis of optical density of the negative control).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 mg (For the solid test item 30 μl of 0.9% NaCl were used to moisten and ensure good contact with the epidermis surface. A piece of nylon mesh was used as a spreading aid).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 0.9 % NaCl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % Sodium Dodecyl Sulfate (SDS)

Duration of treatment / exposure:

20 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of 3 cultures

Value:

ca. 95

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

- 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
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Sample No.	Test item	Time [min.]	% Cell Viability
1 -3	negative control NaCl 0.9 %	20	100.00
7 -9	test item	20	95.28
4 -6	positive control SDS 5%	20	3.12

Interpretation of results:

GHS criteria not met

Executive summary:

An in vitro study for predicting non-specific irritating properties of the test item was conducted according to OECD TG 439. The undiluted test item (30 mg per insert) was applied topically to a reconstructed human epidermis model (RhE; epiCS). The cell viability was determined with 95.28 % for the test item as measured by MTT conversion. It is therefore concluded that the test item is not irritating to skin.

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:

weight of evidence

Study period:

March 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

1981

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS

- Source: dr. Ivanovas gmbH, 7964 Kisslegg im Allgäu/Germany
- Age at study initiation: 12-16 weeks
- Weight at study initiation: 2.3-3.0 kg
- Housing: individually in stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 4 days

- Temperature (°C): 22+- 2
- Humidity (%): 55 +- 10
- Photoperiod (hrs dark / hrs light): 12

Vehicle:

unchanged (no vehicle)

Controls:

other: the right eye remained untreated and served as a control

Amount / concentration applied:

0.1 g/animal and left eye

Duration of treatment / exposure:

3 of 6 rabbits until the eyes were examined and ocular reactions recorded at 1, 24, 48 and 72 hours
in 3 of 6 rabbits approx. 30 seconds after treatment the treated eye was flushed with physiological saline for about one minute.

Observation period (in vivo):

up to 21 days, unless the eye is normal at 72 hours, 7 or 14 days

Number of animals or in vitro replicates:

3/sex

Details on study design:

scoring according to Draize

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

males/not rinsed

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

females/rinsed

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

males/not rinsed

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

mean

Remarks:

females/rinsed

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

males/not rinsed

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

conjunctivae score

Basis:

mean

Remarks:

females/rinsed

Time point:

24/48/72 h

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

males/not rinsed

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

females/rinsed

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

One our after treatment redness (grade 1) was observed in 2/3 males (not rinsed). In 3/3 males (not rinsed) discharge was observed one hour after treatment. No effects were observed in 3/3 females (rinsed) one our after treatment.

Other effects:

There were no systemic intolerance reactions.

Interpretation of results:

GHS criteria not met

Executive summary:

In an eye irritation study with the test substance performed according to OECD 405 on six rabbits (3/sex), the test substance induced no irritation when applied into the rabbit eye mucosa. No acute toxicological signs were observed.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 Jun 2019

Qualifier:

according to guideline

Guideline:

other: EU-method B.69 (Reconstructed human cornea-like epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage.)

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: three-dimensional human cornea model tissue model

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
In principle the EpiOcular™ eye irritation test (EIT) measures the ocular irritation potential of a test item by determination of cytotoxic effects on a reconstructed human cornea epithelium (RhCE) tissue model to discriminate chemicals not requiring classification for eye irritancy (UN GHS No Category) from those requiring classification. The EpiOcular™ EIT is not intended to differentiate between UN GHS Category 1 (serious eye damage) and UN GHS Category 2 (eye irritation). This differentiation would need to be addressed by another tier of a test strategy. As a BCOP has already been conducted and Category 1 can be excluded based on the result, this test can be used to determine, if the substance needs to be classified (Category 2) or not.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The experiment was carried out on the EpiOcular™ RhCE tissue construct (about 0.6 cm² in size; MatTek Corporation, Slovakia).
The RhCE tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s medium (Part#: OCL-200; Lot No.: 30629; Testing date: 8 Oct 2019).

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg test item and 50 µl negative and positive control to the EpiOcular™ tissue surface in duplicate.

Duration of treatment / exposure:

6 hours ± 15 min. at standard culture conditions (5 % CO2, 37 °C, 95 % humidity) followed by a post-soak immersion period of about 25 min in fresh medium

Duration of post- treatment incubation (in vitro):

the tissues were incubated for 18 hours ± 15 min in fresh medium under SCC (37 °C, 5 % CO2, 95 % humidity).

Number of animals or in vitro replicates:

each 2 tissues for the test item, positive and negative controls

Details on study design:

- RhCE tissue construct used, including batch number: The EpiOcular™ RhCE tissue construct consists of 3 viable layers of cells and a non-keratinized surface as recommended by the test guidelines. The cell viability and barrier function as well as sterility of each batch of the RhCE tissue construct used is adequate, as has been demonstrated by the supplier.
RhCE tissue viability in EpiOcular™ EIT is measured by enzymatic conversion of the vital dye MTT by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): 37 ± 2 °C; 6 h exposure, 25 min. post-soak immersion, 18 h post-treatment incubation

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): Killed control and colour control not required.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): each 2 tissues for the test item, positive and negative controls

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): The concentration of formazan was measured by determination of the OD of the isopropanol-extracts in duplicate at 570 nm in an automatic reader of a spectrophotometer (EL808, Bio-Tek; 96 well format, 200 µL).

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: For interpretation of cell viability results the cut-off value distinguishing classified (irritant) from non-classified substances as given in OECD TG 492 was used:
- The test chemical is identified as not irritant and not requiring classification according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60 %.
- The test chemical is identified as irritant and potentially requiring classification according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60 %.

- Positive and negative control means and acceptance ranges based on historical data: The following acceptance criteria determine the validity of an assay:
- mean OD 570 nm negative control (NC) is > 0.8 and < 2.5
- mean relative viability of the positive control (PC) is < 50 % (relative to negative control)
- the difference of viability between the two replicates is < 20 %.

Irritation parameter:

other: cell viability (%)

Run / experiment:

mean

Value:

62

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Killed control: not required
- Colour control not required.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

GHS criteria not met

Conclusions:

The study was conducted under GLP according to OECD guideline 492 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the irritation potential of the test item to the eye in vitro.
As the final test item-treated tissue viability was > 60 % (62 %) relative to negative control, the test item can be characterized as NOT having eye irritating properties.

Executive summary:

The model used is standardized and commercially available. The EpiOcularTM Eye Irritation Test (EIT) was conducted in accordance with OECD 492. The solid test item was applied topically to the RhCE tissue surface in duplicate for 6 hours, followed by an 18 hours post-treatment incubation period.

Cell viability was measured in a spectrophotometer by assessing the extent of MTT (methylthiazole tetrazolium) reduction. The optical density value obtained for the test item was used to calculate the percentage of viability relative to the negative control, which was set at 100 %.

The results of the concurrent negative control (NC, deionized water) and positive control (PC, neat methyl acetate) demonstrated the viability (NC) and sensitivity (PC) of the tissue model. The final mean percent tissue viability recorded for the test item is 62 % (rounded).

According to the results of this study the test item was identified as not requiring classification for eye irritation according to UN GHS (No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available studies no classification according to EU Regulation 1272/2008 is required for skin and eye.