Ammonium benzoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17.03.2017 - 28.06.2017

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

other: not applicable

Details on animal used as source of test system:

no animals used

Justification for test system used:

test system according international accepted Guideline

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number(s): 25802
- Production date: not stated
- Shipping date: not stated
- Delivery date: 28. Mar. 2017
- Date of initiation of testing: 27. Mar 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +- 1 °C
- Temperature of post-treatment incubation (if applicable): 37 +- 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 1 rinsing step, volume not stated
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter: none
- Filter bandwidth: none
- Linear OD range of spectrophotometer: not stated

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tested with MTT assay
- Barrier function: tested with 1.0 % Triton X-100
- Morphology: due to viability test and barrier function test, morphology is assumed
- Contamination: tested for HIV-1 virus, Hepatitis B virus, Hepatitis C virus, Bacteria, yeast and other fungi
- Reproducibility: within the range of historical data

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
possible interference tested in pretests

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive or irritant to skin if the viability is less than 50% of negative control
- The test substance is considered to be non-corrosive and non-irritant to skin if is greater than or equal to 50%

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):24.3, 25.4 and 26 mg

VEHICLE
none

NEGATIVE CONTROL
Dulbecco's Phosphate Buffered Saline
- Amount(s) applied (volume or weight): 30 µL

POSITIVE CONTROL
Sodium dodecyl sulphate
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

1 hour

Duration of post-treatment incubation (if applicable):

41 hours and 25 min

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 1

Value:

81

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 2

Value:

82.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 3

Value:

78.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

80.6

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: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: range of historical values not different from guideline

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Ammonium Benzoate is considred as non-irritant to skin in the Reconstructed humgan Epidermis (RhE) Test Method.

Executive summary:

Three tissues of the human skin model EpiDerm^TM were treated with Ammonium Benzoate for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control, 5% SDS solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 <= mean OD >= 2.8, OD was 2.0. The positive control showed clear irritating effects. Relative absorbance was reduced to 2.7% (required <= 20%).

Variation within the tissue replicates was acceptable (required: <= 18%).

After the treatment with the test item, the relative absorbance value was reduced to 80.6%. This value is above the threshold for skin irritation potential (50%).