Reaction mass of hentriacontan-16-one and pentatriacontan-18-one and tritriacontan-16-one

Endpoint summary

Description of key information

Skin irritation/corrosion:

Two in vitro studies are available.

One in vitro skin irritation study was performed using a human three dimensional epidermal model based on the most recent OECD 439 guidelines (2015). The test item is non-irritant under the experimental conditions.

Another in vitro skin corrosion test was performed to evaluate test item ability to induce skin corrosion using a human skin model according to OECD 431 (2016). The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions.

 

Eye irritation:

Bovine Corneal Opacity and Permeability test (BCOP test) was performed based on the most recent OECD guideline 437.The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.1 after 4 hours of treatment.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-07-17 to 2017-07-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch No.: FK650468
Purity: not specific

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): 26708 kit L and M
- Date of initiation of testing: 17 Jul 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: incubation of 3 minutes: at room temperature, others at 37.0 ± 1.0°C (actual range 36.8 - 37.5°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washed with phosphate buffered saline, the rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: two tissues each for 3-minute exposure, 1-hour exposure, negative and positive controls

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if 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 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

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 32.0 to 35.6 mg

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

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

Duration of treatment / exposure:

3-minute and 1-hour

Irritation / corrosion parameter:

% tissue viability

Value:

86

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The relative mean tissue viability obtained after the 3-minute treatments with the test item

Irritation / corrosion parameter:

% tissue viability

Value:

92

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The relative mean tissue viability obtained after the 1-hour treatments with the test item

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction/Colour interference with MTT: the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8) and the laboratory historical control data range.
- Acceptance criteria met for positive control: The mean relative tissue viability following the 1-hour exposure to the positive control was 6.2%.
- Acceptance criteria met for variability between replicate measurements: In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤11%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In vitro skin corrosion test was performed to evaluate test item ability to induce skin corrosion using a human skin model according to OECD 431 (2016).The possible corrosive potential of test was tested through topical application for 3 minutes and 1 hour.

Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of test item was applied directly on top of the skin tissue.

The test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with test item compared to the negative control tissues was 86% and 92%, respectively. Because the mean relative tissue viability for test item was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, the test item is considered to be not corrosive.

In conclusion, the test item is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2017-08-22 to 2017-08-28

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No.: FK650468
Purity: not specific

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize 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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Tissue batch number(s): 17-EKIN-034
- Date of initiation of testing: 22 Aug 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 15 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): the skin tissues were incubated for 42 hours at 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance
medium until all tissues were dosed and rinsed.

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

NUMBER OF REPLICATE TISSUES: 3 tissues each for test item treatment, negative and positive control.


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive 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-corrosive 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 11.90 to 17.17 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μl 5% SDS

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Irritation / corrosion parameter:

% tissue viability

Value:

117

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item

Other effects / acceptance of results:

- OTHER EFFECTS:
- Colour interference with MTT: no color changes and test item did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 15%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was ≤ 6%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In vitro skin irritation test was performed using a human three dimensional epidermal model based on the most recent OECD 439 guidelines (2015). The possible skin irritation potential of the test item was tested through topical application for 15 minutes.

Skin tissue was moistened with 5 μL of Milli-Q water and at least 10 mg of test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-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 MTT at the end of the treatment.

 

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with test item compared to the negative control tissues was 117%. Since the mean relative tissue viability for test item was above 50% after 15 ± 0.5 minutes treatment test item is considered to be non-irritant.

 

In conclusion, test item is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

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:

From 2017-07-17 to 2017-07-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch No.: FK650468
Purity: not specific

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: from the slaughterhouse
- Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 349 to 368 mg

Duration of treatment / exposure:

240 ± 10 minutes

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- Preparation of Corneas: The isolated corneas were stored in a petri dish with cME (Earle’s Minim um Essential Medium) containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.
- Cornea Selection: After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group

NEGATIVE CONTROL USED
physiological saline

POSITIVE CONTROL USED
20% (w/v) Imidazole solution

APPLICATION DOSE AND EXPOSURE TIME
- Application dose: 750 μL of the negative control and positive control, 349 to 368 mg for test item
- Exposure time: 240 ± 10 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by the diminution of light passing through the cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Value:

>= -1.5 - <= 0.9

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
The corneas were clear after the 240 minutes of treatment with test item. No pH effect of the test item was observed on the rinsing medium.
The corneas treated with test item showed opacity values ranging from -1.6 to 0.8 and permeability values ranging from 0.003 to 0.011.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.1 after 240 minutes of treatment.

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:

In conclusion, since the test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The eye hazard potential of test item was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test) based on the most recent OECD guideline 437.

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of test item was tested through topical application for approximately 240 minutes.  

Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 123 and within two standard deviations of the current historical positive control mean.  It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.1 after 4 hours of treatment.

In conclusion, since test item induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Skin irritation/corrosion:

The test item is non-irritant in the in vitro skin irritation test and not corrosive in the in vitro skin corrosion test.

According to Regulation (EC) No 1272/2008, section 3.2.2.1, this substance is not classified for this endpoint.

 

Serious eye damage/eye irritation:

Not irritation, IVIS=-0.1

According to Regulation (EC) No 1272/2008, section 3.3.2.1, this substance is not classified for this endpoint.