1,2,3-Propanetriol, mono- and diformates

Endpoint summary

Description of key information

Skin irritation/corrosion: corrosive
Eye irritation/corrosion: corrosive 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

28 - 31 Aug 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study. Read-across from analogous substance. For details on read-across please refer to the attached read-across report.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted 13 April 2004)

Deviations:

yes

Remarks:

additional exposure period of 240 min; limit of evaluation criteria at 35% instead of 50%

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Species:

human

Strain:

other: EpiSkin(TM); reconstructed three-dimensional human epidermis

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: SkinEthic Laboratories, Nice, France

TEST METHOD
The EPISKIN(TM) model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen (EPISKIN(TM) Model Kit 0.38 cm²). A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising the main basal, spinous and granular layers and a functional stratum corneum. The procedure followed is based on the recommended EpiSkin(TM) Skin Corrosivity Test protocol INVITTOX No. 118. The test item is applied topically to the straturm corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKIN(TM) model. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control.

ADAPTATION TO CELL CULTURE CONDITIONS
Tissues were transferred into 12-well plates containing 2.2 mL of prewarmed maintenance medium and incubated for 2 days at 37 °C and 5% CO2.

Type of coverage:

open

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with 0.9% w/v sodium chloride solution served as negative controls, positive controls were exposed to glacial acetic acid.

Amount / concentration applied:

TEST MATERIAL: 50 µL

NEGATIVE CONTROL SUBSTANCE: 0.9% (w/v) sodium chloride solution; 50 µL

POSITIVE CONTROL SUBSTANCE: glacial acetic acid; 50 µL

Duration of treatment / exposure:

Test item: 3, 60, and 240 min
Controls: 240 min

Observation period:

Not applicable

Number of animals:

Not applicable
The test was performed in duplicates for each test or control group and treatment period

Details on study design:

TEST SITE
- Area of exposure: 0.38 cm²

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with PBS.
- Time after start of exposure: 3, 60 and 240 min

CELL VIABILITY MEASUREMENTS
2.2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the filled wells. The tissues were incubated for 3 hours at room temperature in a biological safety cabinet ensuring that the plates were protected from light. 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 taken using the EPISKIN(TM) biopsy punch. The epidermis was carefully separated from the collagen matrix and both parts were plated into labelled 1.5 mL micro tubes containing 850 µL of acidified isopropanol. Each tube was plugged mixed thoroughly and stored overnight at room temperature, protected from light to extract formazan crystals out of the MTT-loaded tissues.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured (quantitative viability analysis) at 540 nm using an Anthos 2001 microplate reader.

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of negative controls. Time point: 240 min. Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

92.4

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 3 min. Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

48.4

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

19.7

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item. Time point: 240 min. Reversibility: other: not applicable. (migrated information)

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

5.7

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls. Time point: 240 min. Reversibility: other: not applicable. (migrated information)

Irritant / corrosive response data:

As the mean cell viability of the test item treated tissues was 19.7% compared to the negative control, the test item is considered as corrosive according to the evaluation criteria of this test.

The relative mean tissue viabilty for the positve control was 5.7% relative to the negative control. The mean OD540 for the negative control was 0.157. Thus, the acceptance criteria were satisfied.

Interpretation of results:

corrosive

Remarks:

Migrated information CLP: Skin Corrosive Cat. 1B, H314 Criteria used for interpretation of results: EU

Conclusions:

Ethylene Diformate has been tested positive for skin corrosivity in vitro in the EPISKIN RECONSTRUCTED HUMAN EPIDERMIS MODEL. The testing results are considered to be valid for read-across to the analogous substance 1,2,3-propanetriol, mono- and diformate. For details on read-across please refer to the attached read-across report.

Executive summary:

In a primary dermal irritation study (EPISKIN RECONSTRUCTED HUMAN EPIDERMIS MODEL), reconstructed three-dimensional human epidermis was exposed to 50 µL of ethylene diformate for 3, 60, and 240 minutes on an area of 0.38 cm².  Irritation was scored by the method of cell viability based on mean values. The test item was tested positive for skin corrosivity in vitro. This result is used in a read-across approach in the assessment of 1,2,3-propanetriol, mono- and diformates.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given. Lack of individual data. Read-across from analogous substance. For details on read-across please refer to the attached read-across report.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

not specified

Principles of method if other than guideline:

Treatment of the rabbit eye with differing volumes of the undiluted test substance.

GLP compliance:

no

Species:

rabbit

Strain:

other: albino rabbit, not further specified

Vehicle:

unchanged (no vehicle)

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.02 and 0.005 mL

Duration of treatment / exposure:

single application without washing

Observation period (in vivo):

Reading time point: 18-24 h

Number of animals or in vitro replicates:

5

Details on study design:

SCORING SYSTEM: The individual numerical scores of each eye treated with a given volume or concentration of a chemical were added together and then divided by the number of eyes to obtain the score of the injury caused by the treatment. The level of 5 was selected as representative of severe injury. This figure corresponded to necrosis, visible after staining and covering about 3/4 of the surface of the cornea; or a more severe necrosis covering a smaller area.
See Tables 1 and 2 under "Any other information on materials and methods incl. tables".

TOOL USED TO ASSESS SCORE: fluorescein

Irritation parameter:

other: injury grade

Basis:

mean

Remarks:

out of 5 eyes

Time point:

other: 18-24 h

Score:

5

Max. score:

20

Reversibility:

not specified

Remarks on result:

other: 0.005 mL of the undiluted test substance

Irritation parameter:

other: injury grade

Basis:

mean

Remarks:

out of 5 eyes

Time point:

other: 18 - 24 h

Score:

> 5

Max. score:

20

Reversibility:

not specified

Remarks on result:

other: 0.02 mL of the undiluted test substance

Interpretation of results:

corrosive

Remarks:

Migrated information CLP: Eye damage 1, H318 Criteria used for interpretation of results: EU

Conclusions:

Application of 0.02 and 0.005 mL of the test item leads to necrosis, visible after staining and covering about 3/4 of the surface of the cornea; or a more severe necrosis covering a smaller area. This result is used in a read-across approach in the assessment of 1,2,3-propanetriol, mono- and diformates.

Executive summary:

In a primary eye irritation study, 0.02 and 0.005 mL of ethylene diformate was instilled into the eye of 5 albino rabbits for 18-24 hours. This was a single application without. Irritation was scored by injury grade. Application of 0.02 and 0.005 mL of the test item leads to necrosis, visible after staining and covering about 3/4 of the surface of the cornea; or a more severe necrosis covering a smaller area. In this study, ethylene diformate is corrosive to the eyes of rabbits.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin irritation/corrosion

The potential of ethylene diformate to cause skin corrosion was evaluated using the EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) model in a study performed according to OECD guideline 431 under GLP conditions (Warren, 2012). Duplicate tissues were treated with the undiluted test substance for 3, 60 and 240 min. Concurrent negative (0.9 % sodium chloride solution) and positive (glacial acetic acid) were included for an exposure period of 240 min. Following exposure, the mean tissue viability was determined using the MTT reduction assay and compared to that of the negative control. The relative mean tissue viability after 3, 60 and 240 min exposure was 92.4, 48.4 and 19.7 % of the negative control value. The positive control-treated tissues yielded a relative mean tissue viability of 5.7%. Based on these results and according to the evaluation criteria of the study, the substance is considered to be corrosive to the skin.

Based on the available in vitro data, the substance fulfils the criteria for classification as skin corrosive (Category 1B) according to Regulation (EC) No 1272/2008. Therefore, an in vivo skin irritation study does not need to be conducted in accordance with column 2 of Annex VIII, Section 8.1, of Regulation (EC) No 1907/2006.

The testing results are considered to be valid for read-across to the analogous substance 1,2,3-propanetriol, mono- and diformates. For details on read-across please refer to the attached read-across report.

In vivo eye irritation/corrosion

Ethylene diformate was tested in an in vivo study applying different volumes of the undiluted test substance to rabbit eyes (Carpenter, 1946). In groups of 5 albino rabbits (no further information given), 0.005 to 0.02 mL of the undiluted test substance were applied in a single application without washing. The eyes were observed and reactions were evaluated 18-24 h after application. The individual numerical scores of each eye treated with a given volume or concentration of a chemical were added together and then divided by the number of eyes to obtain the score of the injury caused by the treatment. The level of 5 was selected as representative of severe injury. This figure corresponded to necrosis, visible after staining and covering about 3/4 of the surface of the cornear or more severe necrosis covering a smaller area. The injury grade after application of 0.005 mL and 0.02 mL of the undiluted test substance resulted in a calculated score of ≥ 5 out of five eyes after 18-24 h.

Based on these results, along with the available data on skin irritation/corrosion, the substance is considered to be corrosive and to cause irreversible effects on the eye.

The testing results are considered to be valid for read-across to the analogous substance 1,2,3-propanetriol, mono- and diformates. For details on read-across please refer to the attached read-across report.

Justification for selection of skin irritation / corrosion endpoint:
GLP guideline study on structural related substance

Justification for selection of eye irritation endpoint:
Only one study available.

Effects on skin irritation/corrosion: corrosive

Effects on eye irritation: corrosive

Justification for classification or non-classification

The available data on the skin and eye irritating/corrosive potential of the test item meet the classification criteria for Skin Corrosive Category 1B (H314: Causes severe skin burns and eye damage) and Irreversible Eye Damage Category 1 (H318: causes serious eye damage) according to Regulation (EC) 1272/2008 and for R34 (causes burns) according to Directive 67/548/EEC.