Ammonium benzoate

Endpoint summary

Description of key information

For skin irritation/corrosion one OECD Guideline study available. According to the results, no further studies necessary.

For Eye irritation, two OECD Guideline studies available. According to the results of the study performed first,

a second study was necessary. Based on the results of both studies, no further studies are necessary.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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 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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

tissue 1

Value:

81

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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

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

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

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%).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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)

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

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.

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

Irritation parameter:

other: viability

Run / experiment:

Tissue 1

Value:

0.034

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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 %

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02. June 2017- 08.June 2017

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

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

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
no Test Animals were used

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks' Balanced Salt Solution with 1% Penicillin-Treptomycin solution (Penicillin 100 U/ml, Streptomycin 100 µg/mL) in a suitable cooled container.

Vehicle:

Hank's balanced salt solution

Controls:

yes

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

Test item:
solution with a concentration 20 % Ammonium Benzoate in Hanks' Balanced Salt Solution.

750 µL Test item is applied to the apparatus

Duration of treatment / exposure:

4 h

Observation period (in vivo):

no applicable

Duration of post- treatment incubation (in vitro):

90 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32+-1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 +- 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
visually

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
HBSS

SOLVENT CONTROL USED (if applicable)
HBSS

POSITIVE CONTROL USED
Imidazole solution (20 % in HBSS)
APPLICATION DOSE AND EXPOSURE TIME
750 µL of 20 % solution, 4 hours

TREATMENT METHOD: [closed chamber / open chamber]
Open chamber

POST-INCUBATION PERIOD: yes/no. If YES please specify duration
No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2
- POST-EXPOSURE INCUBATION: 90 min

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry / microtiter plate reader (OD492)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
according to TG

Irritation parameter:

in vitro irritation score

Run / experiment:

mean IVIS of three replicates

Value:

ca. 52.78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

study cannot be used for classification

Conclusions:

Ammonium Benzoate cannot be classified in UN GHS category for eye damage (Category 1). Accordingly the substance cannot be not classified.

Executive summary:

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle which were between

12 and 60 months old.

The test item Ammonium Benzoate was brought onto the cornea of a bovine eye which

had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose

opacity had been measured.

The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the

test item, opacity and permeability values were measured.

HBSS was used as negative control. The negative control showed no irritating effect on

the cornea and the calculated IVIS (in vitro irritancy score) is 0.94.

20% imidazole solution was used as positive control. The positive control induced serious

eye damage on the cornea and was within two standard deviations of the current historical

mean. The calculated IVIS (in vitro irritancy score) is 117.12.

The test item was tested as solution with a concentration of 20 % in HBSS. Under the

conditions of this study, the test item Ammonium Benzoate showed effects on the cornea

of the bovine eye. The calculated IVIS (in vitro irritancy score) is 52.78.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55

induces effects on the cornea, that cannot be classified in UN GHS Category for eye damage

(Category 1). Accordingly the substance cannot be not classified (No prediction can

be made).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with Regulation EC No. 1272/2008, the information on skin irritation/corrosion is conclusive but not sufficient for classification.

In accordance with Regulation EC No. 1272/2008, the information on eye irritation is conclusive. The substance is eye irritating and should be classified as Eye irritant category 2.