4-anilinobenzenediazonium hydrogen sulphate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From March 6th to 29th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Series on Testing and Assessment No 129. 20 Jul 2010. Guidance Document on using Cytotoxicity Tests to Estimate Starting Doses for Acute Oral Systemic Toxicity Tests.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL - ECVAM RECOMMENDATION on the 3T3 Neutral Red Uptake (3T3) Cytotoxicity assay for Acute Oral Toxicity Testing. April 2013.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol n°139. Balb/c 3T3 Neutral Red Uptake Cytotoxicity Assay (3T3 NRU)

GLP compliance:

yes

Test type:

other: in vitro test BALB/c3T3

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-anilinobenzenediazonium hydrogen sulphate

EC Number:

224-756-8

EC Name:

4-anilinobenzenediazonium hydrogen sulphate

Cas Number:

4477-28-5

Molecular formula:

C12H11N3O4S

IUPAC Name:

4-anilinobenzene-1-diazonium hydrogen sulfate

Test material form:

solid: particulate/powder

Test animals

Details on test animals or test system and environmental conditions:

IN VITRO TEST SYSTEM:
- Cell line: murine fibroblasts BALB/ 3T3 cells, clone A31.
- Initial n. of cell: 3 x10^3 cell/100μl/well
- Growth conditions: 37 ± 1 °C, 90 ± 10 % humidity, 5.0 ± 1.0 % CO2/air.

Administration / exposure

Route of administration:

other: In vitro test: application of test item dosing solution on BALB/ 3T3 cells.

Vehicle:

other: cell culture medium .

Details on oral exposure:

VEHICLE
- Justification for choice of vehicle and concentration in vehicle: the test item is a powder and was preliminary tested to evaluate its solubility. The test item resulted to be soluble at 200 mg/ml concentration in culture medium.

Doses:

8 concentrations for test item in the two main experiments: 1.000, 0.465, 0.216, 0.101, 0.047, 0.022, 0.010, 0.005 µg/ml (dilution factor of 1:2.15).
8 concentrations for positive control Sodium Dodecyl Sulfate: 100, 68.03, 46.28, 31.48, 21.42, 14.57, 9.91, 6.74 µg/ml (dilution factor of 1:1.47) .
Six replicates for each concentration.

Details on study design:

MEDIUM:
Dulbecco’s Modified Eagle’s Medium - DMEM , Calf Serum (CS), Penicillin/ Streptomycin solution 100X, Gentamicyn sulfate solution , Sodium bicarbonate , Sodium pyruvate.

REAGENTS:
Trypsin 0,05%/EDTA 0,02% solution, Phosphate buffer without Ca and Mg, Phosphate buffer with Ca and Mg, Ethanol , DMSO, Neutral Red solution 3.3 mg/ml, Glacial Acetic Acid, Distilled water.

CONTROLS:
- Positive control: Sodium Dodecyl Sulfate Solution 10 %
- Vehicle control (VC1 and VC2): 100 % culture medium
- Blank controls: Cxb: test item in the culture medium without cells and VCb: culture medium without cells.

PROCEDURE: Note: the test item was prepared shortly before it is applied and the solutions were kept protected from the light.
# RANGE FINDING TEST: the test item is a powder and was preliminary tested to evaluate its solubility. The test item resulted to be soluble at 200 mg/ml concentration in culture medium. The range finding test was performed to determine the starting doses for the definitive test. Test item was tested in the range finding test at the follow concentrations (w/v): 100000, 10000, 1000, 100, 10, 1, 0.1, 0.01 µg/ml.
# DEFINITIVE TEST: TWO EXPERIMENTS:
Definitive test was performed to determine the IC50 value. The concentration closest to the range finder test IC50 value serves as the midpoint of the concentrations tested in the definitive test. Compared to the range finding test, the definitive test uses a smaller dilution factor for the concentrations tested.
Test item was tested at the follow concentrations (w/v) on the base of the range finding IC50: 1.000, 0.465, 0.216, 0.101, 0.047, 0.022, 0.010, 0.005 µg/ml (dilution factor of 1:2.15).
As a positive control, SDS was diluted in the culture medium and tested at the following concentrations: 100, 68.03, 46.28, 31.48, 21.42, 14.57, 9.91, 6.74 µg/ml (dilution factor of 1:1.47) .
Two definitive tests were perform in different experimental sessions.
- Treatment and exposure: cells were seeded in 96 wells plates (3 x10^3 cell/100μl/well) and allowed to grow for 24 h at 37 ± 1 °C, 90 ± 10 % humidity, 5.0 ± 1.0 % CO2/air. The next day, the medium is replaced with fresh medium supplemented with 8 serial dilutions of the test item. The test was performed in six replicates. After 48 hours of exposition at 37 °C, the viability of the cells is tested by NRU assay.
- NRU assay : cell cultures are washed with phosphate buffer saline (PBS) in order to remove the sample. They are then treated with a Neutral red solution at 25 µg/ml for 3 hours. The excess dye is removed through PBS rinsing, and each well is treated with the extraction solution (EtOH/acetic acid) and mixed using an orbital shaker for 20-45 minutes. The homogenous dye solution obtained is then measured with a spectrophotometer at 540 nm±10nm to quantify the dye uptake in each well.

EXPRESSION OF RESULTS:
Raw data from the microtiter plate reader were be transferred to the Excel® spreadsheet template provided in the ECVAM validation study protocol. A calculation of cell viability expressed as NRU is made for each concentration of the test substance by using the mean NRU of the six replicates values per test concentration (blanks will be subtracted). This value is compared with the mean NRU of all VC values. Relative cell viability is then expressed as percent of untreated VC. An excel template was used for the data analysis and IC50 determination.
The following formula is used to predict the LD50 value:
Log LD50 (mg/kg) = 0.372 log IC50 (µg/ml) + 2.024

EVALUATION CRITERIA:
LD50 > 2000 mg/kg bw: no classification according to the CLP Regulation (EC n.1272/2008) and according to UN GHS

ACCEPTANCE CRITERIA:
1- The left and the right mean of the VC controls do not differ by more than 15 % from the mean of all VC.
2- At least one calculated cytotoxicity value between 0 % and 50 % viability and at least one calculated cytotoxicity value between 51 % and 100 %.
3- The positive control (SDS) IC50 must be within 2.5 standard deviations of the historical mean (i.e. between 15.01 μg/ml and 80.30 μg/ml).

Statistics:

Raw data from the microtiter plate reader were be transferred to the Excel® spreadsheet template provided in the ECVAM validation study protocol.

Results and discussion

Preliminary study:

The preliminary test was performed to evaluate the citotoxicity of the test item (IC50) and to define the concentrations for main test. In the preliminary test 8 concentrations of test item were used (100000, 10000, 1000, 100, 10, 1, 0.1, 0.01 µg/ml). Based on the result of preliminary test the main test was performed starting from 1 µg/ml.

Effect levels

Key result

Dose descriptor:

LD50

Effect level:

ca. 41.54 mg/kg bw

Based on:

test mat.

Remarks on result:

other: LD50 was calculated based on IC50 = 0.0813 μg/ml as mean of two main experiments.

Any other information on results incl. tables

RESULTS:

The test item 4-anilinobenzenediazonium hydrogen sulphate tested on Balb/3T3 cells according to Series on Testing and Assessment No129 and in compliance with Good Laboratory Practice (GLP), showed an IC50 mean value of 0.0813 µg/ml, equivalent to an estimated LD50 value of 41.54 mg/kg. Test item was tested in two different main tests with 8 different concentrations (1.000; 0.465; 0.216; 0.101; 0.047; 0.022; 0.010; 0.005µg/ml). The test item concentration used in the main experiments were chosen based on the results of preliminary dose range finding test

ACCEPTANCE CRITERIA:

All acceptance criteria were fulfilled:

1- The left and the right mean of the VC controls did not differ by more than 15 % from the mean of all VC.

Two VC column difference %: 7.37 % and 1.65 % (both ≤ 15 %).

2- At least one calculated cytotoxicity value was between 0 % and 50 % viability and at least one calculated cytotoxicity value was between 51 % and 100 %.

Definitive test 1: concentrations with cell viability 0 - 50 %: 5; concentration with cell viability 51 - 100 %: 3.

Definitive test 2: concentrations with cell viability 0 - 50 %: 3; concentration with cell viability 51- 100 %: 5.

3- The positive control (SDS) IC50 was within 2.5 standard deviations of the historical mean.

Definitive test 1: IC50 for positive control: 54.2 μg/ml (within 15.01 and 80.30 μg/ml).

Definitive test 2: IC50 for positive control: 78.5 μg/ml (within 15.01 and 80.30 μg/ml).

 

Preliminary dose range finding test

Dose μg/ml	100000.000	10000.000	1000.000	100.000	10.000	1.000	0.100	0.010
Mean cell viability %	-28.8	-5.0	-31.9	-30.7	-30.3	-13.96	37.7	122.9
DS	22.2	4.8	1.9	1.5	1.2	4.8	7.1	17.6

Main experiment 1

Dose μg/ml	1.000	0.465	0.216	0.101	0.047	0.022	0.010	0.005
Mean cell viability %	-2.2	-1.2	0.4	-0.1	24.4	61.2	64.1	83.6
DS	0.7	0.6	1.1	0.9	5.1	1.5	1.8	2.9
IC50= 0.0275 μg/ml

Main experiment 2

Dose μg/ml	1.000	0.465	0.216	0.101	0.047	0.022	0.010	0.005
Mean cell viability %	2.0	1.1	1.1	80.5	105.5	113.9	119.7	118.9
DS	1.2	0.9	0.4	18.9	10.7	8.5	11.3	20.1
IC50 = 0.135 μg/ml

IC50 mean of two experiment: 0.0813 μg/ml

Applicant's summary and conclusion

Interpretation of results:

other: H301 (Acute oral tox cat.3) according to the CLP Regulation (EC n.1272/2008).

Conclusions:

IC50 mean of 2 experiments: 0.0813 μg/ml equivalent to log LD50 (mg/kg) = 0.372 * log IC50 (μg/ml) + 2.024 = 0.372 * log (0.0813) + 2.024 = 0.372 * (-1.089)+ 2.024 = -0.405+2.024 = 1.618, i.e. LD50 = 41.54 mg/kg (around 50 mg/kg bw).

Executive summary:

The purpose of the test is the evaluation of the cytotoxicity of the test item “4-anilinobenzenediazonium hydrogen sulphate according to the Series on Testing and Assessment N.129 and in compliance with Good Laboratory Practice (GLP). The cytotoxicity assay performed in this study was designed to evaluate the cytotoxic potential of the test item solubilised in the medium culture for 48 hours towards the fibroblasts (Cell line BALB/3T3 clone A31 (ATCC-CCL-163)). After 48 hours of incubation with different concentrations of the test item with fibroblasts, viability cell is determined by NRU test. The IC50 value is used in linear regression equation to estimate the oral LD50 value (dose that produces lethality in 50% of the animals tested). The test item “4-anilinobenzenediazonium hydrogen sulphate” did show an estimate LD50 less than 2000 mg/kg (41.54 mg/kg). The positive control showed IC50 value within 2.5 standard deviation of the historical mean. All the acceptance criteria were passed.