Ammonium benzoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6. Mar. 2017 - 27. Jun. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Constituent 1

Reference substance name:

Ammonium benzoate

EC Number:

217-468-9

EC Name:

Ammonium benzoate

Cas Number:

1863-63-4

Molecular formula:

C7H6O2.H3N

IUPAC Name:

ammonium benzoate

Test material form:

solid: crystalline

Details on test material:

- State of aggregation: solid
- Particle size distribution: median particle size D50 = 98 µm
- Mass median aerodynamic diameter (MMAD): not determined
- Geometric standard deviation (GSD): not determined
- Shape of particles: not determined
- Surface area of particles: not determined
- Crystal structure: not determined
- Coating: none
- Surface properties: none known
- Density: 1.26
- Moisture content: ca. 1.5 %
- Residual solvent: none known
- Activation: none
- Stabilisation: none
- Other: none

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 608031
- Expiration date of the lot/batch: 25. Oct. 2018
- Purity test date: not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in the test facility in a closed vessel at room temperature
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: assumed soluble and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: assumend none

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

Test animals / tissue source

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: test system according international accepted Guideline
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live:
The EpiOcular tissue consists of normal, uman-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcular tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50.7 mg and 50.5 mg

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
according to OECD Guideline

- RhCE tissue construct used, including batch number
EpiOcularTM tissue, Batch No.: 23773
- Doses of test chemical and control substances used
ca. 50 mg test substance, 50 µL controls

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
exposure: 6 hours
post-exposure: 48 hours

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
MTT reduction potential and staining properties tested

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
2

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
570 nm

- Description of the method used to quantify MTT formazan
isopropanol extract

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
> 60 % viability: Non eye irritant
=< 60 % viability: Eye irritant

- negative and positive control values within range of historical data

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: viability

Run / experiment:

Tissue 1

Value:

0.034

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: viability

Run / experiment:

Tissue 2

Value:

0.03

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria for negative control: OD between 0.8 and 2.5
- Found value: 1,5

- Acceptance criteria met for positive control: < 50% of negative control
- Found value: 42.1 %

- Range of historical values if different from the ones specified in the test guideline:
For negative control: 1.526 +- 0.249
For positive control: 32.4 % +- 8.3 %

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the test, Ammonium Benzoate is considred as either eye irritant or inducing serious eye damage in the EpiOcular Eye Irritation Test. A differenciation between GHS category 1 or 2 is not possible.

Executive summary:

One valid experiment was performed.

The test item Ammonium Benzoate was applied to a three-dimensional human cornea tissue

model in duplicate for an exposure time of 6 hours.

The solid test item was applied to each tissue.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of

the tissues was evaluated by addition of MTT, which can be reduced to formazan. The

formazan production was evaluated by measuring the optical density (OD) of the resulting

solution.

Demineralised water was used as negative control and methyl acetate was used as positive

control.

The controls showed the following results: After treatment with the negative control, the

absorbance values were within the required acceptability criterion of mean OD > 0.8 and <

2.5, OD was 1.5. The positive control showed clear eye irritating effects, the relative absorbance

value was reduced to 42.1 % (< 50%).

Variation within tissue replicates was acceptable (< 20%).

After treatment with the test item, the mean value of tissue viability was 2.1 %.

This value is below the threshold for eye irritation potential (≤ 60%).

According to the OECD Guideline 492, the EpiOcularTM Eye Irritation Test does not allow

discrimination between eye irritation/reversible effects on the eye (Category 2) and serious

eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing

with other suitable test methods may be required.