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Toxicological information

Acute Toxicity: oral

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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From February 4 to 22, 2019
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:
2019
Report Date:
2019

Materials and methods

Principles of method if other than guideline:
The study was based on:
- Series on testing and assessment no. 129, 20 July 2010,Guidance document on using cytotoxicity tests to estimate starting doses for acute oral systemic toxicity tests
- EURL - ECVAM RECOMMENDATION on the 3T3 Neutral Red Uptake (3T3) Cytotoxicity assay for acute oral toxicity testing, April 2013
- DB-ALM Protocol no. 139, Balb/c 3T3 Neutral Red Uptake Cytotoxicity Assay (3T3 NRU)
GLP compliance:
yes
Test type:
other: in vitro cytotoxicity evaluation

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Administration / exposure

Details on study design:
Range finding
Test item resulted to be soluble at 200 mg/ml in culture medium.
Test item was tested at: 100000, 10000, 1000, 100, 10, 1, 0.1, 0.01 µg/ml.

Definitive tests
Test item was prepared shortly before application and protected from light. Based on the IC50 value in the range finding, test item was tested at: 20000, 13605.44, 9255.4, 6296.19, 4283.12, 2913.69, 1982.1 and 1348.37 µg/ml (dilution factor 1:1.47)
As a positive control, SDS was diluted 1:1.47 at: 100, 68.03, 46.28, 31.48, 21.42, 14.57, 9.91, 6.74 µg/ml.
2 definitive test were performed in experimental sessions.

Treatment and exposure
Cells were seeded in 96 wells plates (2.5-2.6 × 1000 cells / 100 µl/well) and allowed to grow for 24 h at 37 °C ± 1 °C, 90 ± 10 % humidity and 5 ± 1 % CO2 air.
The next day the medium was replaced with fresh medium supplemented by 8 serial dilutions. The test was performed in 6 replicates. After 48 h at 37 °C, the cell viability was tested by NRU assay.

NRU assay
cell cultures were washed with PBS to remove the sample. They are then treated with a neutral red solution at 25 µg/ml for 3 h. The excess dye was removed through PBS rinsing and each well was treated with extraction solution (EtOH/acetic acid) and mixed using an orbital shaker for 20-45 minutes. The homogeneous dye solution wobtained was measure with a spectrophotometer at 540 ± 10 nm to quantify the dye uptake in each well.

Expression of results and interpretation
A calculation of cell viability expressed as NRU was made for each concentration of the test substance by using the mean NRU of the 6 replicates per test concentration (blanks were subtracted). This value was compared to the mean NRU of all values of vehicle controls. Relative cell viability was then expressed as % of untreated vehicle control.
The following formula was used to predict the LD50 value:
log LD50 (mg/kg) = 0.372 logIC50 (µg/ml) + 2.024
An estimated LD50 above 2000 mg/kg is highly predictive of the absence of direct toxic action. Levels below the threshold indicate that further tests are required to assess the real in vivo acute oral toxicity potential of test item.

Acceptability criteria
Negative control: the left and right VC (vehicle controls) do not differ by more than 15 % from the mean of all VC.
Test item: at least 1 calculated cytotoxicity value between 0 % and 50 % viability and at least 1 calculated cytotoxicity value between 51 % and 100 %.
Positive control: IC50 of SDS must be within 2.5 standard deviations of the historical mean.

Results and discussion

Effect levels
Dose descriptor:
LD50
Effect level:
3 233.26 mg/kg bw
Based on:
test mat.
Remarks on result:
other: derived from an IC50 value determined in an in vitro cytotoxicity study

Any other information on results incl. tables

Range finding: IC50 = 11300 µg/ml

Definitive test 1: IC50 = 9780 µg/ml

Positive control SDS: IC50 = 53.3 µg/ml

Definitive test 2: IC50 = 9930 µg/ml

Positive control SDS: IC50 = 53.4 µg/ml

Mean IC50 of 2 experiments: 9855 µg/ml

Estimated LD50 = 3233.26 mg/kg

Detailed data is attached.

All acceptability criteria were passed.

Applicant's summary and conclusion

Interpretation of results:
other: low acute toxicity (data used as part of a weight of evidence evaluation)
Conclusions:
IC50 = 9855 µg/ml
LD50 = 3233.26 mg/kg
Executive summary:

Method

Balb/c 3T3 NRU assay for in vitro cytotoxicity assessment. A preliminary test on solubility in culture medium was run; a range finding test was run to decide test concentrations for the main tests. Two definitive tests were run independently, at concentrations of: 20000, 13605.44, 9255.4, 6296.19, 4283.12, 2913.69, 1982.1 and 1348.37 µg/ml

Each concentration was tested in 6 replicates.

SDS was used as positive control.

Cells were seeded in 96 wells plates and allowed to grow for 24 h at 37 °C, then the medium was replaced with fresh medium supplemented by 8 serial dilutions. After 48 h at 37 °C, the cell viability was tested by NRU assay. In NRU assay, cell cultures were washed with PBS to remove the sample. They were then treated with a neutral red solution at 25 µg/ml for 3 h. The excess dye was removed through PBS rinsing and each well was treated with extraction solution (EtOH/acetic acid) and mixed using an orbital shaker for 20-45 minutes. The homogeneous dye solution obtained was measure with a spectrophotometer at 540 ± 10 nm to quantify the dye uptake in each well.

Results

IC50 = 9855 µg/ml, as mean of 2 experiments

LD50 = 3233.26 mg/kg, estimated from the IC50 value.