Reaction products of cyclododeca-1,5,9-triene and dinitrogen monoxide, distillation overheads

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Remarks:

Turnkey Testing Strategy

Adequacy of study:

weight of evidence

Study period:

Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (4Z,8E)-dodeca-4,8,11-trienal and (4E,8Z)-dodeca-4,8,11-trienal

EC Number:

701-295-5

Molecular formula:

C12 H18 O

IUPAC Name:

Reaction mass of (4Z,8E)-dodeca-4,8,11-trienal and (4E,8Z)-dodeca-4,8,11-trienal

Specific details on test material used for the study:

Test material is the main constituent of the registered substance.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm 200
- Detailed information: surface 0.6 cm²; cultured in Millicells® ∅ 1 cm
- Date of initiation of testing: 16 Aug 2018
- Evaluation of cell viability: 23 Aug 2018
- Tissue model: EPI-200
- Tissue Lot Number: 28646 (Certificate of Analysis see Appendix)
- Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature or 1 h in the incubator
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing procedure: The tissues were washed with sterile PBS 3 min or 1h after start of application treatment

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 3h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: No reference filter

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues (if applicable): Freezing at –20°C
- N. of replicates : 2
- Method of calculation used: Calculation of mean corrected OD570 KC by subtracting the OD570 KC of the NC from the OD570 KC of the test substance

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if complete, e.g. the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL undiluted test substance

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL deionized water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8 N potassium hydroxide solution

Duration of treatment / exposure:

3 min and 1h (for treatments with an viability of ≥ 50% after 3 min)

Number of replicates:

2

Test system

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL undiluted test substance


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL deionized water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL 8 N potassium hydroxide solution
:

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with PBS
- Time after start of exposure: 3min or 1h

OBSERVATION TIME POINTS
- 3 min and 1h

SCORING SYSTEM:
- Method of calculation: The individual tissue OD570 is calculated by subtracting the
mean blank value of the respective microtiter plate from the respective individual tissue OD570 value; KC correction was performend to assess the final relative mean viability

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Exposure period 3min

Value:

104.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Exposure period 1h

Value:

101.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: yes; KC tissues were applied in parallel and the mean viability was corrected

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: The historical control data demonstrate the reproducibility of results and
robustness of the procedures.

Any other information on results incl. tables

Tab. 2: Exposure period 3 min: Individual and mean OD570 values, individual and mean viability values,

standard deviations and coefficient of variation

Exposure period: 3 min
Testsubstance identification			tissue 1	tissue 2	mean	SD	CV [%]
NC	viable tissues	mean OD570	2.071	2.098	2.084
		viability [% of NC]	99.4	100.6	100.0	0.9	0.9
	KC tissues	mean OD570	0.146	0.127	0.137
		viability [% of NC]	7.0	6.1	6.5	0.6	9.8
18/0072-2	viable tissues	mean OD570	2.273	2.231	2.252
		viability [% of NC]	109.1	107.0	108.0	1.4	1.3
	KC tissues	mean OD570KC NC corrected	0.065	0.098	0.081
		viability [% of NC]	3.1	4.7	3.9	1.1	29.2
	Final relative mean viability of tissues after KC correction [% of NC]:				104.2
PC	viable tissues	mean OD570	0.167	0.235	0.201
		viability [% of NC]	8.0	11.3	9.6	2.3	23.8

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in

parallel. The results of the KC tissues indicate an increased MTT reduction (relative mean

viability 3.9% of NC). Thus, for the test substance, the final relative mean viability is given after KC correction.

Tab. 3: Exposure period 1 h: Individual and mean OD570 values, individual and mean viability values,

standard deviations and coefficient of variation

Exposure period: 1 h
Testsubstance identification			tissue 1	tissue 2	mean	SD	CV [%]
NC	viable tissues	mean OD570	2.043	2.084	2.063
		viability [% of NC]	99.0	101.0	100.0	1.4	1.4
	KC tissues	mean OD570	0.116	0.122	0.119
		viability [% of NC]	5.6	5.9	5.8	0.2	3.6
18/0072-2	viable tissues	mean OD570	2.358	2.155	2.256
		viability [% of NC]	114.3	104.4	109.3	7.0	6.4
	KC tissues	mean OD570KC NC corrected	0.199	0.134	0.166
		viability [% of NC]	9.6	6.5	8.1	2.2	27.9
	Final relative mean viability of tissues after KC correction [% of NC]:				101.3
PC	viable tissues	mean OD570	0.122	0.139	0.130
		viability [% of NC]	5.9	6.7	6.3	0.6	9.5

Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in

parallel. The results of the KC tissues indicate an increased MTT reduction (relative mean

viability 3.9% of NC). Thus, for the test substance, the final relative mean viability is given after KC correction.

Tab. 4: Historic control data of NC and PC of corrosion test

Historic Range of NC
OD570
Exposure Time	Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
3 minutes	Jan 2016 - Jul 2018	1.751	0.217	2.184	1.318
60 minutes	Jan 2016 - Jul 2018	1.723	0.225	2.172	1.273
Historic Range of PC
OD570
Exposure Time	Period	Mean OD	SD	Mean + 2 SD	Mean - 2 SD
3 minutes	Jan 2016 - Jul 2018	0.238	0.080	0.398	0.078
60 minutes	Jan 2016 - Jul 2018	0.093	0.019	0.130	0.055
Relative Viability (%)
Exposure Time	Period	Mean %	SD	Mean + 2 SD	Mean - 2 SD
3 minutes	Jan 2016 - Jul 2018	13.6	4.3	22.3	5.0
60 minutes	Jan 2016 - Jul 2018	5.4	1.1	7.6	3.2

Applicant's summary and conclusion

Interpretation of results:

other: no indication of skin corrosion

Conclusions:

No prediction can be made for skin irritation according to GHS criteria based on the results on this in vitro study alone. According to the results of the present study, the substance shows no potential of skin corrosion.

Executive summary:

The objective was to assess the skin irritation and corrosion potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential. Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy:

The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT).

In the SCT the potential of the test substance to cause dermal corrosion was assessed by a

single topical application of 50 μL undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).

For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each.

Due to the intense smell of the test substance, the plates were sealed with an adhesive protective foil immediately after application.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial

dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal

tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 104.2%, and it was 101.3% after an exposure period of

1 hour. Based on the results of the skin corrosion test no skin corrosion potential can be concluded.