Rape oil, reaction products with diethylenetriamine

Endpoint summary

Description of key information

Skin corrosion in vitro: The test item was considered to be non-corrosive to skin because cell viability was determined to be ≥ 50 % after 3 minutes and ≥ 15 % after 60 minutes (OECD 431; EpiDerm Human Skin Model).

 

Skin irritation in vitro: The test item was considered to be non-irritant to skin because the relative mean viability of the test item treated tissues was determined to be 106.2% after the 15-minute exposure period and 42-hours post-exposure incubation (OECD 439; EPISKIN reconstructed human epidermis model).

 

Eye irritation in vitro: The in vitro irritancy score (IVIS) was determined to be 0.2 for the test item. No further testing for eye irritation is required because the IVIS value was ≤ 3 (OECD 437 and EU Method B.47).

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:

key study

Study period:

07 April 2016 to 04 May 2016

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)

Deviations:

yes

Remarks:

various deviations with no impact the integrity or validity of the study (see below)

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008

Deviations:

yes

Remarks:

various deviations with no impact the integrity or validity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

other: neonatal

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 80 mg

Duration of treatment / exposure:

3 minutes or 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

Two

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute exposure

Value:

99.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute exposure

Value:

109.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that negligible interference due to direct reduction of MTT occurred.
- It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results or for reporting purposes.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item did not become coloured.
- This was taken to indicate the test item did not have the potential to cause colour interference.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- Mean OD562 values and viabilities for the negative control, positive control and test item are given in Appendix 1 (attached).
- The relative mean viabilities for each treatment group are shown in the table below.

QUALITY CRITERIA
- The mean OD562 for the negative control treated tissues was 2.003 for the 3-minute exposure period and 1.921 for the 60-Mmnute exposure period. The negative control acceptance criteria were therefore satisfied.
- The relative mean tissue viability for the positive control treated tissues was 4.4 % relative to the negative control following the 60-minute exposure period. The positive control acceptance criterion was therefore satisfied.
- In the range 20 to 100% viability, the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

RELATIVE MEAN VIABILITIES FOR EACH TREATMENT GROUP

Exposure period	Percentage viability negative control*	Percentage viability positive control	Percentage viability test item
3 minute	100	3.6	99.7
60 minutes	100	4.4	109.4

* Mean viability of the negative control tissues is set at 100%

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

GUIDELINE

The purpose of the test was to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Corrosion is directly related to cytotoxicity in the EpiDerm tissue and is determined by reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to formazan by viable cells in the test item-treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item. The study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015) and Method B40bis of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

METHODS

Duplicate tissues were treated with discs of the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

RESULTS

Mean viability of the negative control tissues was set at 100 % and quality criteria for acceptance of results were satisfied. Relative mean viabilities after 3 minutes exposure were determined to be 100 % (negative control), 3.6 % (positive control and 99.7 % (test item). Relative mean viabilities after 60 minutes exposure were determined to be 100 % (negative control), 4.4 % (positive control) and 109.4 % (test item).

 

CONCLUSION

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

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 May 2016 to 09 May 2016

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:

yes

Remarks:

deviations with no impact on purpose or integrity of the study arising from physical state of test substance and preparation of water killed tissues (see below)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

deviations with no impact on purpose or integrity of the study arising from physical state of test substance and preparation of water killed tissues (see below)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Source strain:

other: adult

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Approximately 80 mg

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Three

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Main test

Value:

106.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: relative mean viability of tissues after 15-minute exposure and 42-hour post-exposure incubation

Other effects / acceptance of results:

DIRECT MTT REDUCTION
- The MTT solution containing the test item turned blue which indicated that the test item directly reduced MTT.
- The direct reduction by the test item relative to the negative control value was 10.7 %
- Direct reduction was < 30% relative to the negative control and therefore acceptable.

ASSESSMENT OF COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues.

TEST ITEM, POSITIVE CONTROL ITEM AND NEGATIVE CONTROL ITEM
- The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Appendix 1 (attached). The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Appendix 1.
- The relative mean viability of the test item treated tissues was 106.2 % 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 relative mean tissue viability for the positive control treated tissues was 6.7 % relative to the negative control treated tissues and the standard deviation value of the viability was 0.3 %. The positive control acceptance criteria were therefore satisfied.
- The mean OD562 for the negative control treated tissues was 0.628 and the standard deviation value of the viability was 4.5 %. 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 10.8 %. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was determined to be non-irritant.

Executive summary:

GUIDELINE

The purpose of the test was to evaluate the skin irritation potential of the test item using the EPISKIN 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 formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The study was performed in compliance with OECD Guideline for the Testing of Chemicals No. 439 (Adopted 28 July 2015) and Method B.46.in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

METHODS

Triplicate tissues were treated with discs of the test item for an exposure period of 15 minutes. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item discs were removed from each tissue and 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-labelled 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-labelled 96-well plate. The optical density was measured at 562 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 106.2 % after the 15-minute exposure period and 42-hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.

 

CONCLUSION

The test item was determined to be non-irritant.

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:

28 April 2016

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF BOVINE EYES
- Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Excised eyes were transported to the test facility over ice packs on the same day of slaughter.
- Corneas were prepared immediately on arrival.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Approximately 235 mg

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

Not applicable

Number of animals or in vitro replicates:

Three

Details on study design:

TEST ITEM PREPARATION AND ANALYSIS
- For the purpose of this study the test item was pressed flat and discs were cut out of an appropriate size to cover the entire cornea.
- The discs had an approximate weight of 235mg.

REFERENCE ITEM PREPARATION
- The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.
- The positive control item (imidazole) was used as a 20% w/v solution in 0.9% w/v sodium chloride solution.

PREPARATION OF CORNEAS
- All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

SELECTION OF CORNEAS AND OPACITY READING
- The medium from both chambers of each holder was replaced with fresh complete EMEM.
- A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer (see Annex 1, attached). The average opacity for all corneas was calculated.
- Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

TREATMENT OF CORNEAS
- The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. Approximately 235mg of the solid test item was found to adequately cover the corneal surface. 0.75ml of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
- At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.

APPLICATION OF SODIUM FLUORESCEIN
- Following the final opacity measurement, the permeability of the corneas to sodium fluorescein was evaluated.
- The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL).
- The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes.

PERMEABILITY DETERMINATIONS
- After incubation the medium in the posterior chamber of each holder was decanted and retained.
- Medium representing each cornea (360 μL) was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

HISTOPATHOLOGY
- The corneas were retained after testing for possible conduct of histopathology.
- Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface.
- The cassette was immersed in 10% neutral buffered formalin.

DATA EVALUATION
- Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

OPACITY MEASUREMENT
- The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading.
- These values were then corrected by subtracting the average change in opacity observed for the negative control
corneas.
- The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

PERMEABILITY MEASUREMENT
- The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea.
- The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
- Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

VISUAL OBSERVATION
- The condition of the cornea was visually assessed post treatment.

CRITERIA FOR AN ACCEPTABLE TEST
- Imidazole (20% w/v) was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 66.9 to 101.4.
- Sodium chloride solution (0.9% w/v ) was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2014 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤ 4.1 and for permeability ≤ 0.105.

Irritation parameter:

in vitro irritation score

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL OPACITY AND PERMEABILITY MEASUREMENTS
- Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Appendix 1 (attached).

CORNEAL EPITHELIUM CONDITION
- The condition of each cornea is given in Appendix 2 (attached)
- The corneas treated with the test item were clear post treatment.
- The corneas treated with the negative control item were clear post treatment.
- The corneas treated with the positive control item were cloudy post treatment.

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Scores were determined to be 0.2 for the test item, 1.1 for the negative control and 89.6 for the positive control.

CRITERIA FOR AN ACCEPTABLE TEST
- The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.
- The negative control gave opacity of ≤ 4.1 and permeability ≤ 0.105. The negative control acceptance criteria were therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item score of 0.2 was less than that of the negative and positive controls.

Executive summary:

GUIDELINE

The study was performed in accordance with OECD Guideline for the Testing of Chemicals No. 437 (updated 26 July 2013) “Bovine Corneal Opacity and Permeability Assay”and Method B.47 of Commission Regulation (EC) No 440/2008 to identify whether the test item would induce serious eye damage or not require classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

METHODS

Since it was not possible to formulate a concentration of 20 % w/v, the test item was applied neat to the excised eyes of adult cattle for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

 

RESULTS

In vitro irritancy scores were reported as 0.2 for the test item, 1.1 for the negative control and 89.6 for the positive control.

 

CONCLUSION

The test item score of 0.2 was less than that of the negative and positive controls.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin corrosion in vitro

The key skin corrosion study was performed in compliance with the OECD Guideline for the Testing of Chemicals No 431 In Vitro Skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method (28 July 2015) and Method B.40 bis of Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).Corrosion is directly related to cytotoxicity in the EpiDerm tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The purpose of the test is to evaluate the corrosivity potential of the test item using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes.

 

Duplicate tissues were treated with discs of the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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 2 mL Isopropanol for MTT extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

The relative mean viabilities for each treatment group after an exposure period of 3 minutes were 100 % for the negative control, 3.6 % for the positive control and 99.7 % for test item. The relative mean viabilities for each treatment group after an exposure period of 60 minutes were 100 % for the negative control, 4.4 % for the positive control and 109.4 % for test item. The test item was considered to be non-corrosive to skin because cell viability was determined to be ≥ 50 % after 3 minutes and ≥ 15 % after 60 minutes. Classification for skin corrosivity in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

 

Skin irritation in vitro

The key study for in vitro skin irritation was performed in compliance with OECD Guideline for the Testing of Chemicals No. 439 (Adopted 28 July 2015) and Method B.46 in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). The purpose of the test was to evaluate the skin irritation potential of the test item using the EPISKIN 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 formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

 

Triplicate tissues were treated with discs of the test item for an exposure period of 15 minutes. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the exposure period the test item discs were removed from each tissue and 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-labelled 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-labelled 96-well plate. The optical density was measured at 562 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

The relative mean viability of the test item treated tissues was determined to be 106.2% after the 15-minute exposure period and 42-hours post-exposure incubation. The test item was determined to be non-irritant to skin because percentage tissue viability after exposure and post-treatment incubation was determined to be > 50 %. Classification for skin irritation in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

 

Skin irritation in vivo

Skin irritation does not need to be investigated in vertebrate animals because the test material was demonstrated to be non-hazardous using validated in vitro methods (cell viability ≥ 50 % after 3 minutes and ≥ 15 % after 60 minutes using OECD 431 and percentage tissue viability after exposure and post-treatment incubation > 50 % using OECD 439).

 

Eye irritation in vitro

The key study was performed in accordance with OECD 437 and EU Method B.47 to identify whether the test item would induce serious eye damage or not require classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

 

The test item was applied neat to the excised eyes of adult cattle for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate anin vitroirritancy score (IVIS).

 

In vitro irritancy scores were reported as 0.2 for the test item, 1.1 for the negative control and 89.6 for the positive control.

 

The in vitro irritancy score (IVIS) was determined to be ≤ 3 for the test item and, in accordance with OECD 437 (26 July 2013), no classification is required under the terms of GHS, which is applied in the EU by Regulation (EC) No. 1272/2008 and subsequent amendments. A clear conclusion concerning eye irritation potential was possible using the validated in vitro method and no further testing for eye irritation is required.

 

Eye irritation in vivo

Eye irritation potential does not need to be investigated in vertebrate animals because the test material was demonstrated to be non-hazardous (irritancy score ≤ 3) using the validated in vitro Bovine Corneal Opacity and Permeability (BCOP) assay.

Justification for classification or non-classification

Skin corrosion:The test item was considered to be non-corrosive to skin because cell viability was determined by OECD 431 (28 July 2015) to be ≥ 50 % after 3 minutes and ≥ 15 % after 60 minutes using the EpiDerm Human Skin Model. Classification for skin corrosivity in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

Skin irritation: The test item was determined by OECD 439 (28 July 2015) to be non-irritant to skin because percentage tissue viability after exposure and post-treatment incubation was found to be > 50 % using the EPISKIN reconstructed human epidermis model. Classification for skin irritation in accordance with Regulation (EC) No. 1272/2008 is therefore not required.

Eye irritation: The in vitro irritancy score (IVIS) was determined to be ≤ 3 for the test item in the BCOP assay and, in accordance with OECD 437 (26 July 2013), no classification is required under the terms of GHS, which is applied in the EU by Regulation (EC) No. 1272/2008 and subsequent amendments.