2,4-dichlorobenzyl alcohol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-09-06 to 2018-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010-12-08

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: see remarks

Version / remarks:

Schrage A, Kolle SN, Rey Moreno MC, Norman K, Raabe H, Curren R, van Ravenzwaay B, and Landsiedel R (2011). The Bovine Corneal Opacity and Permeability Test in Routine Ocular Irritation Testing and its Improvement within the Limits of OECD Test Guideline 437. ATLA 39: 37–53.

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: see remarks

Version / remarks:

Kolle SN, Rey Moreno MC, Mayer W, van Cott A, van Ravenzwaay B, Landsiedel R. (2015). The EpiOcular™ Eye Irritation Test is the Method of Choice for the In Vitro Eye Irritation Testing of Agrochemical Formulations: Correlation Analysis of EpiOcular Eye Irritation Test and BCOP Test Data According to the UN GHS, US EPA and Brazil ANVISA Classification Schemes. ATLA 43: 181-198.

Test guideline 5

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

2,4-dichlorobenzyl alcohol

EC Number:

217-210-5

EC Name:

2,4-dichlorobenzyl alcohol

Cas Number:

1777-82-8

Molecular formula:

C7H6Cl2O

IUPAC Name:

(2,4-dichlorophenyl)methanol

Test material form:

solid

Details on test material:

Name of test substance: 2,4-Dichlorbenzylalkohol
Batch identification: DCBA140905
CAS No.: 1777-82-8
Purity: 99.9 area-% (for details see Study code: 16L00070)
Identity: Confirmed (for details see Study code: 16L00070)
Homogeneity: The test substance was homogeneous by visual inspection
Storage stability: The stability under storage conditions over the study period was given.
pH value: Ca. 5
Physical state / color: Solid / white
Storage conditions: Room temperature

Specific details on test material used for the study:

Name of test substance: 2,4-Dichlorbenzylalkohol
Batch identification: DCBA140905
CAS No.: 1777-82-8
Purity: 99.9 area-% (for details see Study code: 16L00070)
Identity: Confirmed (for details see Study code: 16L00070)
Homogeneity: The test substance was homogeneous by visual inspection
Storage stability: The stability under storage conditions over the study period was given.
pH value: Ca. 5
Physical state / color: Solid / white
Storage conditions: Room temperature

Test system

Vehicle:

other: diluted for BCOP using water; undiluted for EpiOcular

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

BCOP:
- Test substance: 750 μL 750 μL 20% (w/v) test-substance preparation (non-surfactant) were applied directly to the epithelial surface of the cornea by using a syringe (open chamber method).
- Negative control (NC): 750 μL of deionized water
- Positive controls (PC): 750 μL Imidazole (CAS No. 288-32-4) 20% (w/v) solution.

EpiOcular:
- Test substance: 50 μL test material was applied undiluted
- Negative control (NC): 50 μL of deionized water
- Positive controls (PC): 50 μL methyl acetate

Duration of treatment / exposure:

BCOP: 4 hours (non-surfactant solids).
EpiOcular: 6 hours

Duration of post- treatment incubation (in vitro):

BCOP - 1.5 hours
EpiOcular - 18 hours

Number of animals or in vitro replicates:

BCOP: The test substance, a negative control (NC; deionized water) and positive controls (Imidazole (CAS No. 288-32-4) 20% (w/v) solution) were applied to three corneas each.
EpiOcular:The test substance, a negative control (NC; deionized water, sterile) and positive controls (Imidazole (CAS No. 288-32-4) 20% (w/v) solution) were applied to two cell cultures each.

Details on study design:

BCOP

TEST SYSTEM
- Test system: isolated bovine cornea
- Source: obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months)
- Supplier: Schlachthof Alzey, Emil Färber GmbH & Co. KG, Robert-Bosch-Strasse 23, 55232 Alzey, Germany

DATA EVALUATION
- In Vitro Irritancy Score (IVIS)
- Permeability Score
- In Vitro Irritancy score (IVIS)
- Histopathological findings

TREATMENT GROUPS
- Test substance
- Negative control (NC): Deionized water
- Positive control (PC): Imidazole (CAS No. 288-32-4) 20% (w/v) solution in deionized water for non-surfactant solid test substances

TEST SUBSTANCE PREPARATION:
- Test-substance preparation: The test-substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a
high-speed homogenizer (Ultra-Turrax) and a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.
- Vehicle: Deionized water
- Reason for the vehicle: Deionized water has been demonstrated to have no adverse
effects on the test system.
- Form of application: 20% (w/v) suspension in deionized water

ACCEPTANCE CRITERIA
1) PC gives an IVIS that falls within two standard deviations of the current historic mean
2) NC responses should result in opacity and permeability values that are not higher than the established upper limits
3) variability between the corneas treated per test substance should be acceptably low

MATERIALS
- Corneal holder: Supplier: BASF SE, Germany
- Incubator: Temperature 32 ± 1°C
- Opacitometer: Kit BASF-OP3.0, BASF SE, Germany
- Spectrophotometer: SunriseTM Absorbance Reader
- Measurement using wavelength of 490 nm

REAGENTS
- Hanks' Balanced Salt Solution with Ca2+ and Mg2+ (HBSS) (Biochrom, Germany) containing Fetal Bovine Serum (FBS) and/or Penicillin/Streptomycin (P/S)
- Eagle’s MEM without phenol red (Biochrom, Germany) containing FBS and P/S
- Eagle´s MEM with phenol red (Biochrom, Germany)
- Sodium fluorescein diluted in DPBS

POST-EXPOSURE TREATMENT
- at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red).
- incubation (1,5 hours)

INCUBATION CONDITIONS
- Temperature: 32°C

MEASUREMENT OF FINAL CORNEAL OPACITY
- visual observations
- opacitometer


EpiOcular

TEST SYSTEM
- Test system: EpiOcularTM model (OCL-200), a three-dimensional, non-keratinized tissue construct composed of normal human-derived, epidermal keratinocytes
- Source: EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts and are commercially available as kits
- Supplier: SMatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

DATA EVALUATION
-The OD570 values determined for the various tissues are measures of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the
NC (percent of control) is used for evaluating whether a test material was an irritant.

TREATMENT GROUPS
- Test substance
- Negative control (NC): Deionized water, sterile
- Positive control (PC): Neat methyl acetate (CAS No.: 79-20-9)
- MTT reduction control (KC): Deionized water, sterile, or test substance

TEST SUBSTANCE PREPARATION:
- Test-substance preparation: The test substance is applied undiluted. Therefore, no preparation of the test substance in a vehicle was performed.

ACCEPTANCE CRITERIA
1) Barrier function and Quality control: ET50 (min) value following exposure to 100 μL of 0.3% Triton X-100 must fall within a range established based on a historical
database of results. Lower acceptance limit: ET50 = 12.2 min Upper acceptance limit: ET50 = 37.5 min
2) Acceptance criteria for the NC: Tissue viability is acceptable if the mean OD570 of the NC is > 0.8. The mean OD570 of the NC should not exceed 2.5.
3) Acceptance criteria for the PC: Methyl acetate used as PC usually leads to a tissueviability of approx. 25%. A viability of < 50% is acceptable.
4) Acceptance criteria for tissue variability: Two tissues were treated under the same conditions. A variability between the two tissues is considered to be acceptable if the relative difference of the viability is < 20%.
5) Acceptance criteria for the KC: The OD570 of the killed control tissues treated as negative control should be ≤ 0.35. The value for direct MTT reduction of a test substance should be ≤ 30% of the NC.

MATERIALS
- Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION) CO2 incubator: Heraeus BBD 6220
- Incubation conditions: 37°C ± 1°C, 5% ± 1% CO2, 90% ± 5% relative humidity
- Spectrophotometer: SunriseTM Absorbance Reader. For the determination of the optical density of colored extracts. Measurement using a filter wavelength 570 nm without reference filter
- EpiOcular™ OCL-200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, containing: 24 OCL-200 tissues (reconstructed cornea): surface 0.6 cm² cultured in Millicells® ∅ 1 cm
- Tissue for MTT reduction control: OCL-200 tissue that is killed by freezing at –20°C

REAGENTS
- Assay medium: OCL-200-ASY assay medium
- MTT diluent: Dulbecco's modified eagle's medium (DMEM) based medium used for diluting MTT (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia / Sigma, Germany)
- Pretreatment / wash buffer: Dulbecco's phosphate buffered saline (PBS), w/o Ca2+, Mg2+ (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, and Biochrom, Germany)
- Detection agent: 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) (MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, and Sigma, Germany), 1.0 mg / mL MTT diluent
- Extracting agent: Isopropanol p.a.

POST-EXPOSURE TREATMENT
- washed with sterile PBS (three times)
- incubation (18 hours)

INCUBATION CONDITIONS
- Temperature: 37°C

MEASUREMENT OF VIABILITY
- OD570 values determined of sample compared to controls

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

BCOP

Run / experiment:

mean value of 3 corneas

Value:

1.5

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: Permeability score

Remarks:

BCOP

Run / experiment:

mean value of 3 corneas

Value:

4.803

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

BCOP

Run / experiment:

mean value of 3 corneas

Value:

73.5

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

other: histopathological score of irritation (HSI)

Remarks:

BCOP

Value:

4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

severe findings

Results 5

Irritation parameter:

other: tissue viability

Remarks:

EpiOcular

Run / experiment:

mean of 2 tissue cultures / % value compared to control

Value:

1.8

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the results of the BCOP and EpiOcular Tests it was concluded that 2,4-Dichlorbenzylalkohol causes ocular corrosion or severe irritation in the in vitro eye irritation test strategy under the test
conditions chosen.