Ammonium benzoate

Endpoint summary

Description of key information

two in vitro studies planned/performed for Endpoint Skin sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16. Mar. 2017 - 31. Mar. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

yes

Remarks:

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is only based on the OECD 442D Guideline.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

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

Details on the study design:

Cell Cultures
For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen
in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees
similar parameters of the experiment and reproducible characteristics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 5 were used. For the experiments
cells of passage 7 was used. After thawing the cells were cultivated in DMEM (9 %
FCS) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Negative Control
DL-Lactic acid
Final concentration: 5000 μM

Positive Control
EGDMA (Ethylene glycol dimethylacrylate)
Final concentration: 120 μM

Solvent Control
DMSO
Final concentration: 1 %

A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concentration
range applicable for experiment I and II. In the CRFT, cytotoxicity was determined
by measuring the cell viability with MTT. A reduction of the viability below 75 % is defined
as a cytotoxic effect.
In the CRFT the following 12 nominal concentrations of the test item were tested:
0.98 μM, 1.95 μM, 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM,
500 μM, 1000 μM, 2000 μM

Dose Selection for Experiment I and II
In accordance with the OECD guideline 442D and the protocol of the BASF SE, the maximum
final test item concentration should be 2000 μM. For a test chemical which has no
defined molecular weight, the final test item concentration 400 μg/mL can also be used.
In the case of a cytotoxic result, the concentrations for experiment I and II should be determined
so that at least one of them is in the cytotoxic range.
Since no cytotoxic reaction was observed in the CRFT the following 12 nominal concentrations
were chosen for experiment I and II: 269 μM, 323 μM, 388 μM, 465 μM, 558 μM,
670 μM, 804 μM, 965 μM, 1157 μM, 1389 μM, 1667 μM, 2000 μM

Experimental Parameters of Experiment I and II

Experiment I and II were performed in the same way. Experiment II serves only to confirm
the results of experiment I.
At the time of seeding the cells were 90 % confluent. The cells were washed twice with
PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized
until the cells detached. The cells were resuspended in medium no. 2. After quantification
the cell suspension was adjusted to 83 000 (±10 %) cells per mL. 120 μL of the cell suspension
(≙ 10 000 cells) were seeded in all wells except H12 (blank) of two clear flat bottom
96 well plates. Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified
atmosphere for 24 h in experiment I and 24 h in experiment II.
For the performance of the viability assay one of the plates was used. The MTT working
solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All
solutions were removed from the wells of the 96 well plate and 200 μL MTT working solution
were added to each well. The plates were incubated for 2 h at 37 ± 1 °C and 5.0 ±
0.5 % CO2 in a humidified atmosphere. Afterwards the solution was removed and 100 μL
of lysis buffer were added to each well. The plate was agitated for 5 min before it was
measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability was
measured by the reduction of the tetrazolium dye MTT (3-(4,5-Dimethyl thiazole 2-yl)-2,5-
diphenyltetrazolium-bromide) (yellow color) to its insoluble formazan (purple color) in living
cells and therefore indicates the amount of living cells. After the measurement of the color
change, the values were transferred in a validated spreadsheet for the calculation of the
viability.
For the performance of the luciferase induction the second plate was used. After the incubation
time all solutions were removed from the cells and 150 μL medium no. 3 was added
to each well. Afterwards 50 μL of the single test item concentrations as well as controls
were added to the cells in triplicates (only for test item concentrations). 12 wells were used
as growth control. 24 wells were used as solvent control, 6 wells were used as negative
control and 5 wells were used as positive control. The arrangement of the substances on
the 96 well plate is demonstrated in figures 15-a and 16-a. The plate was sealed with
breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination
between wells. Afterwards the plate was incubated for 48 h at 37 ± 1 °C in a humidified
atmosphere containing 5.0 ± 0.5 % CO2.
For the evaluation of the luciferase expression the medium was removed from the wells
and the cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Afterwards 100 μL per
well of a Lysis buffer were given to the cells and incubated for 5 min at room temperature.
During this process the plate was slightly moved. The Steady-Glo® Reagent was prepared
by mixing Steady-Glo®-Substrate with Steady-Glo®-Buffer. After lysis 100 μL Steady-Glo®
Reagent were added to each well and the plate was shaken slowly for 5 min at room temperature.
Then, 160 μL per well were transferred to a white flat bottom 96 well plate and
the luminescence was measured for 2 seconds using a luminometer.

Positive control results:

The positive control induced a clear effect with an induction value of 5.7 fold in comparison to the solvent control.

Key result

Run / experiment:

other: all tested concentrations, Experiment I

Parameter:

other: relative Induction of Luciferase

Remarks:

compared to Solvent control

Value:

0.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: all tested concentrations, Experiment I

Parameter:

other: Relative Viability [%]

Value:

100.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: all tested concentrations, Experiment II

Parameter:

other: relative Induction of Luciferase

Remarks:

compared to Solvent control

Value:

0.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: all tested concentrations, Experiment II

Parameter:

other: Relative Viability [%]

Value:

102.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported,
the test item Ammonium Benzoate possesses no sensitizing potential.

Executive summary:

This in vitro study was performed to investigate the sensitizing potential of Ammonium

Benzoate, by using the LuSens cell line.

The assay was performed in a cytotoxicity range finder test (CRFT) and two independent

experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed

to detect a potential cytotoxic effect of the test item. Based on the results of this

test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (2000 μM) was chosen with

regard to the cytotoxic reaction in the CRFT. Furthermore, a geometric series (factor 1.2)

of eleven dilutions was prepared.

DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth

control. Furthermore, Lactic acid (5000 μM) was used as negative control and EGDMA

(120 μM) as positive control.

The evaluated experimental points and the results are summarised chapter 8, page 19f.

No substantial and reproducible dose dependent increase in luciferase induction above 1.5

fold was observed in both experiments up to the maximal concentration of the test item.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06. November - 11. November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Name Ammonium Benzoate
Batch no. 608031
Appearance colorless crystals/powder
Composition Monoconstituent
Purity 99.27 %
Homogeneity homogeneous
Expiry date 25. Oct. 2018
Storage Room Temperature: (20 ± 5 °C)
CAS No. 1863-63-4
EINECS-No. 217-468-9
Chemical Class not stated
Volatility unknown
pH-value 6-7 at 50 g/L
Molecular weight 139.154 g/mol
Stability H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

Details on the study design:

Peptides with ≥ 95 % purity were used.
Cys-Peptide (Cysteine)
Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol)

Lys-Peptide (Lysine)
Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol)

Positive controls were treated identically as the test item. The following positive controls were used:
•Cinnamaldehyde (CAS 104-55-2, food grade 99.1 %, ) was used as 100 mM solution in acetonitrile for the Cys-peptide.
•2,3-Butanedione (CAS 431-03-8, 99.4 %,) was used as 100 mM solution in acetonitrile for the Lys-peptide

Incubation
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 22.1 h for Lys-peptide and 24.3 h for Cys-Peptide.
Measurements were performed using a HPLC method

Acceptance criteria
a) The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
b) The mean peptide depletion value for the positive control 2,3-butanedione should be 10 % - 45 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
c) The maximum standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion

Positive control results:

Mean Depletion of Positive Control for Cys Peptide was 75.41 % with SD 1.92 %.
Mean Depletion of Positive Control for Lys Peptide was 36.66 % with SD 2.69 %.

Key result

Run / experiment:

other: Mean of three determinations

Parameter:

other: Cys-peptide depletion (%)

Value:

0.16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Mean of three determinations

Parameter:

other: Lys-peptide depletion (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: mean of all determinations

Parameter:

other: Mean depletion of both peptides (%)

Value:

0.08

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Acceptance of results:
a) The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and ≤ 14.9 %, respectively, for the Cys-peptide.
b) The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide.
c) The maximum standard deviation for the test item replicates was < 14.9 % for the percent cysteine depletion for the test item.
The maximum standard deviation for the test item replicates was < 11.6 % for the percent lysine depletion for the test item.

Interpretation of results:

study cannot be used for classification

Conclusions:

For Ammonium Benzoate the DPRA predicition is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model.

Executive summary:

The study was performed in order to evaluate the reactivity of the test itemAmmonium Benzoatetowards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in water was incubated 24 ± 2 h at 25 °C together with cysteine and lysine peptides, respectively, and the peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured in parallel.

The peptide depletion values after incubation are shown in below Table:

Cys-peptide depletion [%]	Lys-Peptide depletion [%]	Mean peptide depletion [%]
0.16	0.00	0.08

 

 

The DPRA predicition is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

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 sensitisation is conclusive but not sufficient for classification.