N,N'-diphenyl-p-phenylenediamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 Feb 2016 to 22 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Version / remarks:

26 July 2013

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:

8 December 2010

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-diphenyl-p-phenylenediamine

EC Number:

200-806-4

EC Name:

N,N'-diphenyl-p-phenylenediamine

Cas Number:

74-31-7

Molecular formula:

C18H16N2

IUPAC Name:

N,N'-diphenyl-p-phenylenediamine

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test-substance No.: 16/0045-1
- Batch identification: 20150925
- Purity test date: 82.5 %
- Physical state / colour: solid / grey to brown

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 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/or a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
20% (w/v) suspension in de-ionized water

Test animals / tissue source

Species:

cattle

Strain:

other: bovine

Details on test animals or tissues and environmental conditions:

TISSUE MODEL
- Test system: Isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
- Supplier: Schlachthof Mannheim, Schlachthofstr. 21, 68165, Mannheim, Germany

MATERIALS AND TECHNICAL EQUIPMENT
- 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 Ca++ and Mg++ (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

Test system

Vehicle:

water

Remarks:

de-ionized

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- 750 μL of the 20% (w/v) test-substance preparation (non-surfactant) was applied directly to the epithelial surface of the cornea using a syringe (open chamber method).

CONTROLS
For the control tissues 750 μL of de-ionized water (negative control, NC) or 750 μL of 20% (w/v) solution of imidazole in de-ionized water (positive control, PC), were applied into the anterior chamber using a pipette.

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
- After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 544 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups. Each corneal holder was uniquely identified with a number on the chambers.

APPLICATION DOSE AND EXPOSURE TIME
- Before application, the medium in the anterior chamber was removed using a syringe.
- The 20% (w/v) test-substance preparation could not be applied with a pipette. Therefore 750 μL of the 20% (w/v) test-substance preparation (non-surfactant) was applied directly to the epithelial surface of the cornea using a syringe (open chamber method). For the control tissues 750 μL of de-ionized water (negative control, NC) or 750 μL of 20% (w/v) solution of imidazole in de-ionized water (positive control, PC), were applied into the anterior chamber using a pipette.
- The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids).

TREATMENT METHOD
- Controls: Closed chamber
- Test material: Open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- The NC and PC were removed after the exposure period from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
- Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (5 mg/mL for solid test substances) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined. An aliquot was diluted 1:5 in Eagle’s MEM (without phenol red) and measured analogously (PC, only).

SCORING SYSTEM
In Vitro Irritancy Score (IVIS)

ACCEPTANCE CRITERIA
- A study is considered acceptable if the PC gives an IVIS that falls within two standard deviations of the current historic mean.
- The NC responses should result in opacity and permeability values that are not higher than the established upper limits.
- Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. In cases of borderline results in the first testing run, a second testing run should be considered (but not necessarily required), as well as a third one in case of discordant mean IVIS results between the first two testing runs. In this context, a result in the first testing run is considered borderline if the predictions from the 3 corneas were non-concordant, such that:
* 2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,
* 1 of the 3 corneas gave a discordant prediction from the mean of all 3 corneas, AND the discordant result was >10 IVIS units from the cut-off threshold of 55.

DECISION CRITERIA
See 'Any other information on materials and methods incl. tables'

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean corrected opacity change

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

fluorescein leakage

Run / experiment:

Mean corrected OD490

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean IVIS score

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not valid

Positive controls validity:

valid

Other effects / acceptance of results:

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

In combination with EpiOcular test (OECD 492)