Bis(8-hydroxyquinolinium) sulphate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Remarks:

In vitro NRU cytotoxicity assay on Balb/3T3 cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: In vitro NRU cytotoxicity assay on Balb/3T3 cells

Version / remarks:

EURL – ECVAM 2013: Recommendation on the 3T3 NRU Assay for Supporting the Identification of
Substances Not Requiring Classification for Acute Oral Toxicity
EURL - ECVAM 2011: Follow-up study on the predictive capacity of the 3T3 Neutral Red Uptake
cytotoxicity assay to correctly identify substances not classified for acute oral toxicity under the EU
CLP system (LD50 > 2 000 mg/kg) Final Study Report1

Principles of method if other than guideline:

In vitro NRU cytotoxicity assay on Balb/3T3 cells is predictive for the identification of substances outside the toxicity classification threshold (LD50 values > 2000mg/kg) in Acute Oral Toxicity studies. LD50 calculation for the prediction of in vivo toxicity through in vitro NRU vitality testing. The test is performed on mono-layer murine fibroblasts. Range finding + main test.
The test can discriminate between substances that do not require CLP toxicity classification from potentially harmful and toxic samples. The in vitro toxicity test consist in a NRU analysis (Neutral Red Uptake). The NR dye is normally absorbed by cells and accumulates in lysosomes. Dead or dying cells have damages lysosomes and cellular membranes, and are therefore unable to detain the dye. The presence of cytotoxic substances can either damage the cells or impair their replication, resulting in a proportional and measurable reduction of the amount of detained dye. The colorimetric quantification of the dye’s uptake therefore provides an accurate evaluation of the effects of the sample administered to cell cultures.
According to studies performed by the European Center for the Validation of Alternative Methods (ECVAM,Recommendation on the 3T3 NRU Assay for Supporting the Identification of Substances Not Requiring Classification for Acute Oral Toxicity), the toxicity level observed in NRU tests is predictive of the results of in vivo studies. The test specifically identifies substances with a LD50 (50% lethality dose in acute oral toxicity tests on mice) above 2000 mg/Kg.

GLP compliance:

no

Remarks:

The in vitro NRU cytotoxicity assay on Balb/3T3 cells is a non validated test (non OECD), thus GLP compliance is not required

Test type:

other: In vitro NRU cytotoxicity assay on Balb/3T3 cells

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Bis(8-hydroxyquinolinium) sulphate

EC Number:

205-137-1

EC Name:

Bis(8-hydroxyquinolinium) sulphate

Cas Number:

134-31-6

Molecular formula:

C9H7NO.1/2H2O4S

IUPAC Name:

bis(8-hydroxyquinolinium) sulphate

Test material form:

solid: particulate/powder

Remarks:

yellow powder

Test animals

Species:

other: The test is carried out on a murine fibroblasts (Balb/3T3 cells, clone A31)

Strain:

other: The test is carried out on a murine fibroblasts (Balb/3T3 cells, clone A31)

Sex:

not specified

Administration / exposure

Details on oral exposure:

The test is carried out on a murine fibroblasts (Balb/3T3 cells, clone A31). Cells are cultured in DMEM containing 5% CS and antibiotics.
The cells were seeded in 96-well plates (starting concentration: approximately 3000 cells per well) and expanded in standard medium and culture conditions (37°C, 5% CO2) for 24 hours.

Doses:

The tested sample was diluted in low serum medium at a starting concentration defined by range finding test at 0,002 mg/ml. Seven progressive 1:1.47 dilutions were then prepared.

Control animals:

other: The positive control is Sodium Lauryl Sulfate

Details on study design:

Range finding test:
The cells were seeded in 96-well plates and expanded in standard medium and culture conditions (37°C, 5% CO2) for 24 hours.
The tested sample was diluted in low serum medium at a starting concentration of 0,01 mg/ml. Seven progressive 1:10 dilutions were then prepared.
Sample exposition was performed removing the standard medium and replacing it with the eight progressive sample dilutions. Each sample dilution was tested in triplicate. Blanks consisting in the same concentrations of each sample and control without cells were prepared. Cells cultured in pure dilution medium have been
used as a negative control. All cell cultures (samples, positive and negative controls) and the blanks were incubated in standard conditions for 48 hours.

Main test:
The cells were seeded in 96-well plates (starting concentration: approximately 3000 cells per well) and expanded in standard medium and culture conditions (37°C, 5% CO2) for 24 hours. The tested sample was diluted in low serum medium at a starting concentration defined by range finding test at 0,002mg/ml. Seven progressive 1:1.47 dilutions were then prepared. The positive control (Sodium Lauryl Sulfate) was diluted to a starting concentration of 100μg/ml and seven progressive 1:1.47 dilutions were prepared as for the sample. Sample exposition was performed removing the standard medium and replacing it with the eight progressive
sample dilutions. Each sample dilution was tested in triplicate. Blanks consisting in the same concentrations of each sample and control without cells were prepared. Cells cultured in pure dilution medium have been used as a negative control. All cell cultures (samples, positive and negative controls) and the blanks were incubated in standard conditions for 48 hours.

Neutral Red Uptake (NRU) cell vitality assay:
After 48hours incubation cell viability has been evaluated by NRU (neutral red uptake assay).
Cell cultures are washed with phosphate buffer saline (PBS) in order to remove the sample. They are then treated with a 20% neutral red solution in dilution terrain for 3 hours. The excess dye is removed through PBS rinsing, and each cell culture is treated with an extraction solution and mixed using an orbital shacker for 45 minutes. The homogenous dye solution obtained is then measured with a spectrophotometer to quantify the dye uptake in each culture.

Statistics:

Negative control: OD ≥ 0,2
Standard deviation must be ≤ 18%
The means of the two columns of controls cannot vary by more than 15%
In the main test at least one calculated cytotoxicity value > 0% and ≤ 50% viability and least one calculated cytotoxicity value > 50% and < 100% viability should be present. Exception: if a test has not or has only one point between 0 and 100% and smallest practical dilution factor (i.e. 1.21) was used and all other test
acceptance criteria were met, then the test is acceptable.

Positive control (CP):
The IC50 value for the positive control (SLS) should be within two standard deviations of the historical mean; The positive control’s standard deviation must be ≤ 18%

Results and discussion

Effect levels

Effect levels 1

Sex:

not specified

Dose descriptor:

other: IC50

Effect level:

0.001 other: mg/ml

Based on:

test mat.

Remarks:

main test

Effect levels 2

Sex:

not specified

Dose descriptor:

other: IC50

Effect level:

51.74 other: mcg/ml

Based on:

other: SLS control

Effect levels 3

Sex:

not specified

Dose descriptor:

LD50

Effect level:

84.18 mg/kg bw

Based on:

test mat.

Mortality:

According to the calculated IC50 value and the related studies by ECVAM and OECD, a predictive evaluation can be performed in order to calculate LD50, the dose that would cause a 50%mortality in vivo in murine acute oral toxicity tests.
The calculation is performed according to the following formula: Log LD50 (mg/kg) = 0.372 log IC50 (μg/mL) + 2.024

Applicant's summary and conclusion

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

LD50 = 84.18 mg/Kg bw. According to this result and to the CLP Regulation (EC n. 1272/2008), the substance can be classified in Toxicity Class 3.

Executive summary:

The test item was tested in the "In vitro NRU cytotoxicity assay on Balb/3T3 cells".

The results shown an IC50 value of 0.0005 mg/mL, equivalent to an estimated LD50 value of 84.18 mg/kg bw.

According to this result and to the CLP Regulation (EC n. 1272/2008), the substance can be classified in Toxicity Class 3.