1,3-Propanediaminium, 2-hydroxy-N1,N1,N1,N3,N3-pentamethyl-N3-[3-[(2-methyl-1-oxo-2-propen-1-yl)amino]propyl]-, chloride (1:2)

Endpoint summary

Description of key information

- Not irritating to skin (GLPs and OECD 431 and OECD 439 compliant studies)

- No irritating to eyes (GLPs and OECD 437 compliant study)

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:

From 08 October to 27 November 2015

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:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: CY50561001
- Expiration date of the batch: 24/04/2016
- Purity test date: 65.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature 15-25°C, below 70 RH%
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water. Stability not checked

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none (The test item was applied in its original form.)
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

Test system:

human skin model

Remarks:

EPISKIN-SM is a three-dimensional human epidermis model.

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Source strain:

not specified

Details on animal used as source of test system:

Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period.

Justification for test system used:

The EPISKIN-SM model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM model
- Tissue batch number(s): 15-EKIN-040
- Production date: Unknown
- Shipping date: Unknown
- Delivery date: Unknown
- Expiry date: 12 October 2015
- Date of initiation of testing: 08 October 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one, with approximately 25 mL PBS solution.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT stock solution= 3 mg/mL ; MTT final concentration (working solution) = 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14
- Wavelength: 570 nm.
- Filter: unknown
- Filter bandwidth: unknown
- Linear OD range of spectrophotometer: unknown

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkin-SM test kits used in the present study).

NUMBER OF REPLICATE TISSUES: Two epidermis units were used for each test or control materials.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.
- Fresh tissues
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : two
- Method of calculation used: The mean optical density (measured at 570 nm) of these tissues was determined as 0.009, Non Specific Colour % was calculated as 1.0% (see Table 1). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA:
If both disks have mean viability of ≥35% = Non Corrosive
If both disks have mean viability of <35% = Corrosive (at the corresponding incubation period)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 μL
- Concentration (if solution): N/A (The test item was applied in its original form (it was supplied in a form of 65.4% solution), no formulation was required.)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): N/A

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
- Concentration (if solution): N/A

Duration of treatment / exposure:

4 hours (±10 min)

Duration of post-treatment incubation (if applicable):

N/A

Number of replicates:

Two epidermis units were used for each test or control materials.

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 89.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
- Direct-MTT reduction: no
- Colour interference with MTT: As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.009, Non Specific Colour % was calculated as 1.0% (see Table 1). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary. As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Negative and positive controls as well as variability between replicate measurements were within acceptable limits and therefore the study was considered to be valid.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the two negative control tissues was in the recommended range (0.889).
- Acceptance criteria met for positive control: The positive control treated tissues showed 0.9% viability demonstrating the proper performance of the assay.
- Acceptance criteria met for variability between replicate measurements: The difference of viability between the two test item-treated tissue samples in the MTT assay was 9.3%.

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Diquat, the mean cell viability was 89.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN model test with Diquat, the results indicate that the test item (tested in a form of 65.4% solution) is not corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test of Diquat test item was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (detailed in 3.6. section). The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKIN (two units) were treated with Diquat test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with Diquat, the mean cell viability was 89.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with Diquat, the results indicate that the test item (tested in a form of 65.4% solution) is not 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:

From 21 October to 17 December 2015

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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: CY50561001
- Expiration date of the batch: 24/04/2016
- Purity test date: 65.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature 15-25°C, below 70 RH%
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water. Stability not checked

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none (The test item was applied in its original form.)
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

Test system:

human skin model

Remarks:

EPISKIN-SM is a three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum.

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Source strain:

not specified

Details on animal used as source of test system:

Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994)

Justification for test system used:

The EPISKIN-SM model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM model
- Tissue batch number(s): 15-EKIN-042
- Production date: Unknown
- Shipping date: Unknown
- Delivery date: Unknown
- Expiry date: 26 October 2015
- Date of initiation of testing: 21 October 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature (25.2-25.3°C)
- Temperature of post-treatment incubation (if applicable): 37°C in an incubator with 5% CO2.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: one, with PBS solution.
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT stock solution= 3 mg/mL ; MTT final concentration (working solution) = 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14
- Wavelength: 570 nm.
- Filter: unknown
- Filter bandwidth: unknown
- Linear OD range of spectrophotometer: unknown

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkin-SM test kits used in the present study).

NUMBER OF REPLICATE TISSUES: Three replicates were used for the test item and for each controls.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : As the test item was coloured, one additional test item-treated tissue was used for the non specific OD evaluation.
- Fresh tissues
- Procedure used to prepare the killed tissues (if applicable): N/A
- N. of replicates : one
- Method of calculation used: The optical density (measured at 570 nm) of the additional test item-treated tissue was 0.032, Non Specific Colour % was calculated as 3.7%. This value was below 5%, therefore additional data calculation was not necessary

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA:
The test substance is considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal equal (≤) to 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20 μL
- Concentration (if solution): N/A (applied as such)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): N/A (PBS applied as such)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 5% (w/v) SDS solution

Duration of treatment / exposure:

15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

N/A

Number of replicates:

Three replicates were used for the test item and for each controls. As the test item was coloured, one additional test item-treated tissue was used for the non specific OD evaluation.

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 84.1

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:
- Visible damage on test system: no
- Direct-MTT reduction:
- Colour interference with MTT: As no colour change (yellow colour) was observed after three hours of incubation of the test item in MTT working solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
As the test item was coloured, one additional test item-treated tissue was used for the non specific OD evaluation. The optical density (measured at 570 nm) of this tissue was 0.032, Non Specific Colour % was calculated as 3.7%. This value was below 5%, therefore additional data calculation was not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Negative and positive controls as well as variability between replicate measurements met the acceptability criteria, therefore the study was considered to be valid.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD value of the three negative control tissues was in the recommended range (0.880). Standard deviation of the viability results for negative control samples was 4.3.
- Acceptance criteria met for positive control: The positive control treated tissues showed 11.1% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.6.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 6.8.

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Diquat, the mean relative viability was 84.1% compared to the negative control value. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN model test with Diquat, the results indicate that the test item is non-irritant to skin (No Category).

Executive summary:

An in vitro skin irritation test of Diquat test item was performed in a reconstructed human epidermis model. EPISKIN-SM is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified by spectrophotometry.

PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with Diquat, the mean cell viability was 84.1% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with Diquat, the results indicate that the test item is non-irritant to skin (No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 January to 26 February 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

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: CY50561001
- Expiration date of the batch: 24/04/2016
- Purity test date: 65.4%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water. Stability not checked

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none (The test item was applied in its original form.)
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

Species:

other: bovine cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA,
Saint-Pierre-sur-Dives, France.
- Number of animals: unknown
- Characteristics of donor animals (e.g. age, sex, weight): bovine cattle were up to 12 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.
- Time interval prior to initiating testing: As the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.
- indication of any existing defects or lesions in ocular tissue samples: Any eyes with defects were discarded.
- Indication of any antibiotics used: see above (penicillin/streptomycin (100 units/100 μg/mL final))

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL (± 8 μL)
- Concentration (if solution): as such

Duration of treatment / exposure:

10 minutes (± 30 seconds)

Duration of post- treatment incubation (in vitro):

2 hours (± 10 minutes)

Number of animals or in vitro replicates:

The test item, the negative and the positive control were tested on three corneas each.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
A careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light.
The tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
The corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 μg/mL final) at room temperature. The corneas were used within a maximum of 24 hours.
Storage of the corneas: as the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

QUALITY CHECK OF THE ISOLATED CORNEAS:
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded.

NUMBER OF REPLICATES:
The test item, the negative and the positive control were tested on three corneas each.

NEGATIVE CONTROL USED: 0.9% Sodium Chloride (NaCl)

SOLVENT CONTROL USED (if applicable): N/A

POSITIVE CONTROL USED: Absolute Ethanol

APPLICATION DOSE AND EXPOSURE TIME: 750 μL (± 8 μL) for 10 minutes (± 30 seconds)

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: four times.

- POST-EXPOSURE INCUBATION: 2 hours (± 10 minutes)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.

- Corneal permeability: After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution.
As the test item is a non-surfactant liquid, the concentration of the fluorescein solution was 4 mg/mL.
Before use, the fluorescein solution was validated. For this purpose, the solution of fluorescein was diluted in cMEM in order to obtain a 5 μg/mL solution and the Optical Density at a wavelength of 490 nm (OD490 nm) of this dilution was measured. As the value obtained was between 0.850 and 0.940 (see Table 2), the
fluorescein solution was validated.
For each series of three corneas, a chronometer started from the fluorescein solution application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes).
At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).
Any solutions giving an OD490 nm beyond the linear range of the spectrophotometer was diluted in cMEM and measured again. The corresponding OD490 nm was multiplied according to the dilution factor used.

- Others (e.g, pertinent visual observations, histopathology): After permeability determination, the corneas were removed from the holders and observed for opaque
spots, other irregularities and any separation of the epithelium.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: YES

Irritation parameter:

in vitro irritation score

Value:

ca. 0

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:
- Visible damage on test system: No notable opaque spots or irregularities were observed on negative control and test item-treated corneas.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline:

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, Diquat, was identified as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, Diquat, to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The design of this study was based on the guideline OECD Guideline 437 and the study was performed in compliance with CiToxLAB France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.

A single experiment was performed using three corneas for each treated series (test item, positive control and negative control).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item (undiluted), the negative and positive controls were evaluated in a single experiment using a treatment time of 10 minutes and using the closed-chamber method. At the completion of the treatment period, all formulations were removed from the front opening of the anterior chamber and the epithelia were rinsed.

The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed.

After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C.

At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities.

No notable opaque spots or irregularities were observed on each test item-treated cornea.

All acceptance criteria were fulfilled. The study was therefore considered as valid.

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0.

As the mean IVIS was ≤ 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

In conclusion, under the experimental conditions of this study, the test item, Diquat, was identified as a test chemical not

requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two Klimisch score 1 studies are available to assess the skin corrosive and irritating potential and were used as key studies study:

In the first one, performed according to OECD guideline no. 431 and in compliance with GLPs, the possible corrosive potential of Diquat was tested through topical application for 4 hours on a human three dimensional epidermal model (EPISKIN-SM). Following exposure with Diquat, the mean cell viability was 89.3% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive.

In the second study, performed according to OECD guideline no. 439 and in compliance with GLPs, the possible irritating potential of Diquat was tested through topical application for 15 minutes on a human three dimensional epidermal model (EPISKIN-SM). Following exposure with Diquat, the mean cell viability was 84.1% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. 

One Klimisch score 1 study is available to assess the eye corrosive and irritating potential of the registered substance and was used as key study:

In this study, performed according to OECD guideline no. 437 (BCOP test) and in compliance with GLPs, Diquat was applied for 10 minutes as it is on isolated bovine corneas. No notable opaque spots or irregularities were observed on each test item-treated cornea. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0.

As the mean IVIS was ≤ 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage

Justification for classification or non-classification

In an in vitro skin corrosion (OECD 431) and in vitro skin irritation (OECD 439) tests performed using a reconstructed human epidermis model,

the mean relative cell viability following exposure with the registered substance was above the threshold of 35% and 50% compared to the negative control, respectively, indicating that the substance is not corrosive nor irritant to skin.

In addition, in a Bovine Corneal Opacity and Permeability test (OECD 437), the mean In Vitro Irritancy Score was equal to 0 indicating that the substance is not corrosive nor irritant to eyes.

In conclusion the registered substance is not classified for skin and eye irritation/corrosion according to CLP and GHS-UN regulations.