Dipropoxymethane

Endpoint summary

Description of key information

In vitro skin corrosion and irritation tests have been performed according to OECD TG 431 and 439 (EpiSkin), respectively. Based on the observed results, propylal is not classified regarding skin corrosion/irritation hazard.

In vivo eye irritation test has been performed according to OECD TG 405. The CLP criteria to clasify propylal for seriuos eye damage (cat 1) / eye irritation (cat 2) are not met.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 31 January 2018 Experimental completion date 19 February 2018

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Dipropoxymethane (CAS no. 505-84-0)
CAS Number: 505-84-0
Batch: 1711221700R
Purity: 99.93%
Physical state/Appearance: Clear colorless liquid
Expiry Date: 22 November 2019
Storage Conditions: Approximately 4 oC in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: EpiSkin Laboratories, Lyon, France
Date received: 13 February 2018
EpiSkinTM Tissues (0.38cm2) lot number: 18-EKIN-007
Maintenance Medium lot number: 18-MAIN3-006
Assay Medium lot number: 18-ESSC-006

Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.
Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 μL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.
Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 μL of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.
Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
MTT Loading/Formazan Extraction (Day 3)
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. The test item was used as supplied.
10 μL of DPBS. The negative control item, Dulbecco’s Phosphate Buffered Saline (DPBS), was used as supplied.
10 μL of SDS 5%. The positive control item, Sodium dodecyl sulphate (SDS), was prepared as a 5% w/v aqueous solution. The positive control was formulated within 2 hours of being applied to the test system.

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:

mean

Value:

84.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Direct MTT Reduction
The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.

Assessment of Color Interference with the MTT endpoint
The solution containing the test item was colorless. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item
The relative mean viability of the test item treated tissues was 84.8% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.

Quality Criteria
The test was repeated due to a failure to meet the assay acceptance criteria. The relative mean tissue viability for the positive control treated tissues was 8.5% relative to the negative control treated tissues and the standard deviation value of the viability was 5.3%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.820 and the standard deviation value of the viability was 8.1%. 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 8.0%. The test item acceptance criterion was therefore satisfied.

CONCLUSION
The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Interpretation of results:

other:

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 570 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viability of the test item treated tissues was 84.8% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was classified as non-irritant. The following classification criteria apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2008-04-29 to 2008-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Adopted 13 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

EPISKIN model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN
- Tissue batch number(s): 08-EKIN-016

REMOVAL OF TEST MATERIAL AND CONTROLS
At the end of treatment, all residual test item and chemical controls were removed by thoroughly rinsing epidermis units with 25 ml of sterile phosphate buffered saline solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2.2 ml of 0.3 mg/ml MTT
- Incubation time: 3 hours +/- 5 minutes
- Spectrophotometer: Labsystems Multiskan RC spectrophotometer with auto calibration (Finland)
- Wavelength: 550 nm

NUMBER OF REPLICATE TISSUES: three tissue replicates were used for each treatment (exposure time), including 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 35%, if the viability after 3 minutes exposure is greater than or equal to 35% and the viability after 60 minutes exposure is less than 35%, or if the viability after 60 minutes exposure is greater than or equal to 35% and the viability after 240 minutes exposure is less than 35%.
- The test substance is considered to be non-corrosive to skin if the viability after 240 minutes exposure is greater than or equal to 35%

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 (26.3 µl/cm)

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µl

Duration of treatment / exposure:

3, 60 and 240 minutes

Number of replicates:

Three tissue replicates were used for each treatment (exposure time), including controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 240 minutes

Value:

> 35

Negative controls validity:

valid

Positive controls validity:

valid

Table 1. Mean optical density (at 550 nm) values and viabilities for the negative control item, positive control item and test item

Tissue	Exposure period	Optical density	Mean optical density	Standard deviation	Relative mean viability (%)
Negative control	240 minutes	0,937	1,023	0,079	100,00
		1,042
		1,091
Positive control	240 minutes	0,039	0,046	0,006	4,46
		0,047
		0,051
Test item	3 minutes	0,948	0,965	0,070	94,27
		0,904
		1,042
	60 minutes	0,840	0,752	0,118	73,49
		0,798
		0,618
	240 minutes	0,379	0,458	0,078	44,79
		0,461
		0,535

Interpretation of results:

GHS criteria not met

Conclusions:

According to the obtained results (cell viabilities never below 35% for tested exposure times) and reporting to the classification criteria, the test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test was to evaluate the skin corrosion potential of the test item using the EPISKIN^TM reconstructed human epidermis model after treatment periods of 3, 60 and 240 minutes.

The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method

Triplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 500 µl acidified isopropanol for MTT extraction. At the end of the formazan extraction period the optical density was measured at 550 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities of the test item treated tissues were 94.27, 73.49 and 44.79% after exposure periods of 3, 60 and 240 minutes, respectively.

Conclusion

The test item was considered to be non-corrosive to the skin.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2010-03-22 to 2010-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted 24 April 2002

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: breeding farm VELAZ s.r.o., Kolec u Kladna, Czech Republic, RCH CZ 21760152
- Age at study initiation: 4-5 months
- Weight at study initiation: 3.4-3.7 kg
- Housing: Conventional animal room – individually in metallic cages
- Diet (e.g. ad libitum): Standard pelleted diet TM-MAK 1 for rabbits and guinea pigs ad libidum (producer: Ing.Mrkvicka Miroslav – Vyroba krmnych smesi, Mlyn Kocanda, 252 42 Jesenice u Prahy)
- Water (e.g. ad libitum): Drinking tap water ad libitum (quality corresponding to the Regulation No.: 252/2004 Czech Coll. of Law)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C, permanently monitored
- Humidity (%): 30 – 70%, permanently monitored
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 2010-03-22 to 2010-03-29

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): test item (0.1 g) was used in delivered form

Duration of treatment / exposure:

At 24 hours after application the eye was irrigated with water

Observation period (in vivo):

The eyes were examined at 1, 24, 48 and 72 hours after application, then once daily. After recording the observations at 24 hours, the eyes of rabbit were examined with the aid of fluorescinein and the ophthamoscopy. The grades of ocular reaction for single animal (observation of conjunctivae, cornea and iris) were recorded at each examination.

Number of animals or in vitro replicates:

3 (2 males, 1 female)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): the eye was irrigated with water
- Time after start of exposure: at 24 hours after application

SCORING SYSTEM: according to the grading system given in Method B.5 Acute Toxicity: Eye Irritation/Corrosion
Cornea:
0 - No ulceration or opacity
1 - Scattered or diffuse areas of opacity; details of iris clearly visible
2 - Easily discernible translucent area;details of iris slightly obscured
3 – Nacrous area, no details of iris visible, size of pupil barely discernible
4 – Opaque cornea, iris not discernible through the opacy
Maximum possible: 4

Iris:
0 - Normal
1 – Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia; or injection; iris reactive to light (a sluggish reaction is considered to be an effect)
2 – Hemorrhage, gross destruction, or no reaction to light
Maximum possible: 2

Conjunctivae:
0 - Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris) normal
1 - Some blood vessels hyperaermic (injected)
2 - Diffuse, crimson colour, individual vessels not easily discernible
3 - Diffuse beefy red
Maximum possible: 3

Chemosis:
0 - Normal no alterations
1 - Some swelling above normal
2 - Obvious swelling with partial eversion of lids
3 - Swelling with lids about half closed
4 - Swelling with lids more than half closed
Maximum possible: 4

TOOL USED TO ASSESS SCORE: fluorescein and ophthalmoscopy

Irritation parameter:

cornea opacity score

Basis:

animal: #7

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

cornea opacity score

Basis:

animal: #8

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

cornea opacity score

Basis:

animal: #9

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

iris score

Basis:

animal: #7

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: no effect

Irritation parameter:

iris score

Basis:

animal: #8

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: no effect

Irritation parameter:

iris score

Basis:

animal: #9

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: no effect

Irritation parameter:

conjunctivae score

Basis:

animal: #7

Time point:

24 h

Score:

2

Max. score:

3

Reversibility:

fully reversible within: 4d

Irritation parameter:

conjunctivae score

Basis:

animal: #7

Time point:

other: 48/72h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 4d

Irritation parameter:

conjunctivae score

Basis:

animal: #8

Time point:

24 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 48h

Irritation parameter:

conjunctivae score

Basis:

animal: #9

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 5d

Irritation parameter:

chemosis score

Basis:

animal: #7

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

chemosis score

Basis:

animal: #8

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Irritation parameter:

chemosis score

Basis:

animal: #9

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: no effect

Other effects:

No symptoms of systemic toxicity were observed in the animals during clinical observation in the test period and no mortality occured.
Clinical observation and body weight are depicted in Table 1 (Any other information on results incl. tables).

Table 1. Clinical observation and body weight were recorded in the table below:

Rabbit No.	Clinical observation	Body weight (in kg)
		prior to application	at termination
7	No changes	3.50	3.70
8	No changes	3.40	3.60
9	No changes	3.70	3.90

Conclusions:

Examination of eye irritation after single application demonstrated, that the test substance was slightly irritating for eye of rabbit, but all signs of irritation disappeared till 5th day afer exposure.

Executive summary:

The test substance, propylal, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed).

The test was performed according to the Method B.5 Acute Toxicity: Eye Irritation/Corrosion. Council Regulation (EC) No.440/2008, published in O.J. L 142, 2008.

The test was performed initially using one animal (No. 7). Because a corrosive effect was not observed in initial test, the response was confirmed using two additional animals (No. 8 and No. 9).

The following changes were observed on eye at 1 hour after application: conjunctivae - diffuse, crimson colour, individual vessels not easily discernible; chemosis – some swelling above normal in all rabbits. In one rabbit (No. 9) lacrimation was observed at 1 hour after application. Conjunctivae – diffuse, crimson colour, individual vessels not easily discernible or some blood vessels hyperaemic (injected) were observed in all rabbits at the 24 hours after application. Conjunctivae – some blood vessels hyperaemic (injected) were observed in two rabbits at the 48 and 72 hours after application. Changes on the conjunctivae in one animal (No. 9) persisted to the 4th day after application. On the 5th day no alterations appeared. No clinical signs of systemic intoxication were detected.

Examination of eye irritation after single application demonstrated, that the test substance, propylal, was slightly irritating for eye of rabbit, but all signs of irritation disappeared till 5th day after exposure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available