2,2',2''-nitrilotriethanol citrate

Endpoint summary

Description of key information

Based on all available data, the substance is not considered to be irritating to the skin and eye.

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:

weight of evidence

Study period:

19 Apr 2017 to 24 Apr 2017

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:

28 July 2015

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: 1184 ZITRONENSÄURE, TEA-SALZ
- Source of test material: Zschimmer and Schwarz GmbH + Co. KG.
- Batch: SEALS 2016-198-001
- Purity/Composition: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stable under storage conditions until: 03 January 2018 (expiry date)
- Appearance: Clear colourless liquid (determined by Charles River Den Bosch)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- No correction was made for the purity/composition of the test compound

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured

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
- Background: The EPISKIN Small Model is a three-dimensional human epidermis model, which consists of adult human derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum
- Tissue batch number: 17-EKIN-016
- Source: SkinEthic Laboratories, Lyon, France.
- Date of initiation of testing: 19 April 2017
- Expiration date: 24 April 2017

TEST FOR THE INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed. The test item was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model. Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test item did not interfere with the MTT endpoint.

TEST SYSTEM SET UP
On the day of receipt the tissues were transferred to 12-well plates and pre-incubated with pre-warmed Maintenance Medium for 3.5 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

ENVIRONMENTAL CONDITIONS
All incubations, with the exception of the test item incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 82 - 91%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.0 - 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

APPLICATION/TREATMENT OF THE TEST ITEM
The test was performed on a total of 3 tissues per test item together with negative and positive controls. 25 μL of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were 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. Subsequently the skin tissues were incubated for 42 hours at 37°C.

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labelled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
- The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
- The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

INTERPRETATION
A test item is considered irritant in the skin irritation test 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.
A test item is considered non-irritant in the in vitro skin irritation test 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:

The liquid test item was applied undiluted (25 μL) directly on top of the tissue

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:

Irritation test: 15 minute exposure

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS
- The test item did not interfere with the MTT endpoint.
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met.

Interpretation of results:

GHS criteria not met

Remarks:

In combination with skin corrosion results (OECD 431)

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

3 Apr 2017 to 7 Apr 2017

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:

29 July 2016

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142

Version / remarks:

31 May 2008

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: 1184 ZITRONENSÄURE, TEA-SALZ
- Source of test material: Zschimmer and Schwarz GmbH + Co. KG.
- Batch: SEALS 2016-198-001
- Purity/Composition: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Test item storage: At room temperature
- Stable under storage conditions until: 03 January 2018 (expiry date)
- Appearance: Clear colourless liquid (determined by Charles River Den Bosch)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- No correction was made for the purity/composition of the test compound

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: cultured

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 (EPI-200)
- Background: The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Lot no.: 25748
- Source: MatTek Corporation, Ashland MA, U.S.A.

EXPERIMENTAL DESIGN
- Test for the interference of the test item with the MTT endpoint: A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.
- Test for Colour Interference by the Test Item: The test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 50 μL of the test item or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
- Test for Reduction of MTT: The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μL of the test item was added to 1 mL MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple color change or a blue / purple precipitate was observed.

TEST SYSTEM SET UP
- Tissues: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL DMEM.
- DMEM (Dulbecco’s Modified Eagle’s Medium): Supplemented DMEM, serum-free supplied by MatTek Corporation.
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.
- Environmental conditions: All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 - 92%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.7 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

APPLICATION/TREATMENT OF THE TEST ITEM
- The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well.
- The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 1.5 hours at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3- minute exposure to the test item and two for a 1-hour exposure. Fifty μL of the undiluted test item was added into the 6-well plates on top of the skin tissues.
- For the negative and positive controls, 2 tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues were treated with 50 μL 8N KOH (positive control) for both the 3-minute and 1-hour time point.
- After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
The DMEM was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
- The absolute mean OD570 of the two tissues of the negative control should reasonably be within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8)
- The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.

INTERPRETATION
A test item is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
- In addition, a test item considered non-corrosive (viability ≥ 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
The table in ‘Any other information on materials and methods incl. tables’ presents the data interpretation and optional sub-categorisation in case a test item will be corrosive.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

CONTROLS
- Negative control: 50 μL Milli-Q
- Positive control: 50 μL 8N KOH

TEST ITEM
Undiluted (50 μL) directly on top of the tissue.

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test: 3 minute exposure

Value:

76

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Corrosion test: 1 hour exposure

Value:

73

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS
- The test item did not interfere with the MTT endpoint.
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met.

Interpretation of results:

GHS criteria not met

Remarks:

In combination with skin irritation results (OECD 439)

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:

24 Feb 2017 to 28 Feb 2017

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:

July 26, 2013

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identification: 1184 ZITRONENSÄURE, TEA-SALZ
- Source of test material: Zschimmer and Schwarz GmbH + Co. KG.
- Batch: SEALS 2016-198-001
- Purity/Composition: UVCB
- Stable under storage conditions until: 03 January 2018 (expiry date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Test item storage: At room temperature

Species:

cattle

Strain:

other: bovine

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Test system: Bovine eyes were used as soon as possible after slaughter.
- Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- 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 AND CONCURRENT CONTROLS
750 µL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea.

TEST ITEM PREPARATION
A correction factor is not applicable for the BCOP test, therefore no correction was made for the purity of the test item. The test item was tested neat.

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

PREPARATION OF 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.
- The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) 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 AND OPACITY READING
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 (BASF-OP3.0, BASF, Ludwigshafen, Germany). 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.

TREATMENT OF CORNEAS AND OPACITY MEASUREMENTS
Due to a technical error, the first experiment was aborted and a repeat assay was performed. The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
OPACITY MEASUREMENT
- The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity = [I(0) / I - 0.9894] / 0.0251
With I(0) the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
- The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
- The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

APPLICATION OF SODIUM FLUORESCEIN
- Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
- The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

PERMEABILITY DETERMINATIONS
- After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
- The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

INTERPRETATION
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
- Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in the table in ‘Any other information on materials and methods incl. tables’.

Irritation parameter:

cornea opacity score

Run / experiment:

mean opacity

Value:

-1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein leakage

Run / experiment:

mean permeability

Value:

0.001

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

mean in vitro irritation score

Value:

-1.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- The corneas were clear after the 10 minutes of treatment with the test item.
- A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.

Table. Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity^1	Mean Permeability^1	MeanIn vitroIrritation Score^1, 2
Negative control	-1.0	0.006	-0.9
Positive control (Ethanol)	18.5	3.048	64.2
Test item	-1.5	0.001	-1.4

¹ Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

² In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD₄₉₀value).

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation/corrosion

Since a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential, the test substance (CAS no. 29340-81 -6) was tested in two in vitro assays. The ability of the test substance (CAS 29340-81-6) to induce skin irritation was tested on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM)). The possible skin irritation potential of the test item was tested through topical application for 15 minutes. The study procedures were based on the most recent OECD and EC guidelines. The batch the test substance was a clear colourless liquid. The test item was applied undiluted (25 μL) 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 the test item compared to the negative control tissues was 96%. Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant. The positive control had a mean cell viability of 8.8% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 14%, indicating that the test system functioned properly.

The ability of the test substance to induce skin corrosion was tested on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test substance was tested through topical application for 3 minutes and 1 hour. The study procedures were based on the most recent OECD and EC guidelines. The batch of the test item was a clear colourless liquid. The test item was applied undiluted (50 μL) directly on top of the skin tissue. The positive control had a mean relative tissue viability of 6% after the 1-hour exposure. 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. In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 17%, indicating that 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 the test item compared to the negative control tissues was 76% and 73%, respectively. Because the mean relative tissue viability for the 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 substance is non-irritant and not corrosive in the in vitro skin tests under the experimental conditions used.

 

Eye irritation

The eye hazard potential of the test substance (CAS no. 29340-81-6) was evaluated by measuring its ability to induce opacity and increase permeability in an isolated bovine cornea. The eye damage potential of the test item was tested through topical application for 10 minutes. The study procedures were based on the most recent OECD guideline. The batch of the test item was a clear colourless liquid. The test item was applied as it is (750 µL) directly 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 (Ethanol) was 64 and was 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 -1.4 after 10 minutes of treatment.

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

Justification for classification or non-classification

Based on the results of the available studies, classification is not warranted for skin and eye irritation in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.