[No public or meaningful name is available]

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August 01, 2017 to August 09, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Non-Regulatory Method. The test uses cultured human dermal fibroblasts in animal product-free culture, Neutral Red Uptake (NRU) method and a prediction model, based on the GHS classification system for acute toxicity

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: OECD Guideline 129: Guidance document on using cytotoxicity tests to estimate starting doses for acute oral systemic toxicity tests

Deviations:

not specified

Principles of method if other than guideline:

The Neutral Red Uptake (NRU) assay is used to determine cell viability as an indicator of acute toxicity. Neutral Red (a weak cationic dye), penetrates cellular membranes, entering cells via non-ionic diffusion and accumulates intracellularly in lysosomes. Viable cells take up and retain the Neutral Red (NR) dye, while damaged or dead cells do not, therefore, the Neutral Red Uptake (NRU) assay can be employed as a direct measure of cell viability, using membrane integrity as the measured endpoint. Incorporated NR is released from the cells using a solubilisation solution. The absorbance of the NR is quantified using a spectrophotometer.

GLP compliance:

yes (incl. QA statement)

Test type:

other: Neutral Red Uptake (NRU) Cytotoxicity Test Using Human Dermal Fibroblasts in Xeno-Free Culture Conditions

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C16-18 and ethoxylated C16-18 , phosphates (20 moles ethoxylation)

Molecular formula:

C18H39O4P1 (monoester representative, i.e., mono- C18 PSE) C56H115O24P1 (ethoxylated monoester representative, i.e., mono- C16 AE20 PSE) C32H67O4P1 (diester representative, i.e., di- C18 PSE) C112H227O44P1 (ethoxylated diester representative, i.e., di- C16 AE20 PSE)

IUPAC Name:

Alcohols, C16-18 and ethoxylated C16-18 , phosphates (20 moles ethoxylation)

Test material form:

solid

Test animals

Species:

other: Cultured human dermal fibroblasts in animal product-free culture

Strain:

other: Not Applicable

Sex:

not specified

Details on test animals or test system and environmental conditions:

Neonatal human dermal fibroblast cultures - “Xeno-Free” (HDFn-XF) were obtained commercially as cryopreserved primary cells (Lifeline Cell Technology, Carlsbad, USA). They were originally derived from donor tissue with informed consent for the tissue to be used for research purposes, in adherence with the Human Tissue Act (UK) 2004. Xeno-free culture medium and sub-culture reagents (Lifeline Cell Technology, Carlsbad, USA) were free of animal-derived components, providing a fully human cell culture system. Test system was extensively QC tested, by the manufacturer, for a range of parameters including viability upon thawing from cryopreservation, proliferation rate, morphology and sterility (absence of bacteria, fungal growth and mycoplasma). They were also demonstrated to be negative for HIV-1, HIV-2, HBV and HCV.

Administration / exposure

Route of administration:

other: Refer "Details on oral exposure"

Vehicle:

other: Serum-Free culture medium

Details on oral exposure:

1) Method of administration of test substance
A single application of 8 concentrations of the test substance (n=6) was applied in cell culture medium (dilution factor of 2 for the range finding experiment, 1.5 for the main experiments). The top concentration was previously determined by solubility testing.
Range finding experiment (µg/mL): 2000-1000-500-250-125-62.5-31.3-15.6
Main experiments (µg/mL): 750, 500, 333.3, 222.2, 148.1, 98.8, 65.8, 43.9

2) Method of administration of reference substances:
a) Positive control: Sodium dodecyl sulphate (SDS) (Lot number: SLBL1461V, Expiry date: June 2020).
Concentration tested: 100, 83.3, 69.4, 57.9, 48.2, 40.2, 33.5, 27.9 μg/mL in cell culture medium (n = 6, dilution factor of 1.2)
b) Negative control: Fibrolife serum-free culture medium (Lot number: 05685, Expiry date: 31 Aug 17 for RFE, ME3, Sep 18 for ME1. 08 Sep 17 ME2)
A single application of culture medium was applied as the negative control (n=12).

3) Exposure times of test substances and reference substances:
The cells were incubated with the test or reference substance for 24 ± 1h, at 37°C / 5% CO2, 95% RH (Relative Humidity) followed by NRU measurements

Doses:

Range finding experiment (RFE): 2000, 1000, 500, 250, 125, 62.5, 31.3, and 15.6 µg/mL (dilution factor 2)
Main experiment (ME): 750, 500, 333, 222, 148, 98.8, 65.8, and 43.9 µg/mL (dilution factor 1.5)
As per OECD guidance document 129, an initial range finding experiment (RFE) was performed with a range of concentrations based on the outcome of the solubility test (dilution factor of 2 was used) to determine a top concentration for three main experiments (ME) allowing the determination of a more accurate IC50.

No. of animals per sex per dose:

6 replicates for test substance and positive control
12 replicates for negative control

Control animals:

other: culture medium

Details on study design:

Overview

Preliminary testing: Determination of the top concentration by solubility testing

Range finding experiment (RFE): To determine a top concentration for the main experiment.

Main experiment (ME) x 3:
Day 1: Seeding cells (1 x 96-well plates for RFE; 3 x 96-well plate for ME).
Day 2: 24 h after seeding, apply test and reference substances for 24 ± 1h
Day 3: Evaluate the Neutral Red Uptake

Statistics:

Data Analysis for this study were performed following XCellR8 SOP L0064: “Neutral Red Uptake (NRU) Cytotoxicity Test Using Human Dermal Fibroblasts in Xeno-Free Culture Conditions”, using XCellR8 Form F0058: Acute Toxicity Analysis Spreadsheet v01, for processing. This is a Microsoft Excel workbook (created during the project funded by Innovate UK (project number 131726) and validated in-house in August 2017, containing formulae to process the raw data as per SOP L0064. The final data output is a percentage viability value for cells exposed to the test substance relative to the negative control and the IC50 value.

Results and discussion

Preliminary study:

As per OECD guidance document 129, an initial range finding experiment (RFE) was performed with a range of concentrations based on the outcome of the solubility test (dilution factor of 2 was used) to determine a top concentration for three main experiments (ME) allowing the determination of a more accurate IC50 (i.e. the concentration at which a decrease in cell viability of 50% was observed). Based on solubility data, top concentration used in the RFE was 2mg/mL (2000µg/mL).

Effect levels

Key result

Dose descriptor:

other: IC50

Remarks:

the time taken to reduce cell viability to 50% of the negative control

Effect level:

ca. 77.7 - ca. 289.9 other: µg/mL

Based on:

test mat.

Remarks on result:

other: Equivalent predicted LD50: 300-2000 mg/kg bw

Remarks:

Potential EU CLP classification: Category 4

Other findings:

The IC50 value obtained in all experiments were between 10-1000 µg/mL, therefore, the test substance was classified as GHS Category 4 “Harmful if swallowed”, suggesting a low acute toxicity potential.

Any other information on results incl. tables

Results

Solubility results: The solubility was first determined following OECD guidance document 129 to determine the top concentration for the RFE:

 

Tier 1: 200 mg/mL in cell culture medium - Not soluble

Tier 2: 20 mg/mL in cell culture medium - Not soluble

Tier 3: 2 mg/mL in cell culture medium - Soluble

Therefore, the top concentration used in the range finding experiment (RFE) described here after was 2 mg/mL (2000 µg/mL).

Range finding experiment

An initial Range finding experiment (RFE) was performed with a top test substance concentration of 2000 µg/mL (2 mg/mL) and a dilution factor of 2.

A positive control plate was run in parallel, to validate the assay with a top concentration of 100µg/mL and a dilution factor of 1.2.

 

Positive control - RFE

PC-RFE	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	100.0	83.3	69.4	57.9	48.2	40.2	33.5	27.9	0.0
% of Negative Control	86.6%	-0.1%	-0.4%	-0.3%	-0.1%	3.4%	58.2%	21.1%	45.9%	113.4%
SD	12.0%	0.7%	0.6%	1.0%	1.3%	1.8%	45.4%*	15.7%	12.2%	8.5%
% CV	13.83%	-576%	-143%	-351%	-1691%	52.23%	77.98%	74.33%	26.63%	7.45%

Table 1: Cell viability measurements 24 h± 1 h after application of positive control (SDS). NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8: *SD value of the concentration 6 (40.2 µg/mL) is above 15% (45.4%), therefore C6 value (58.2%) was removed for IC₅₀ calculation.

The calculated IC₅₀ was 27.9 µg/mL, which is in the range of the historical data obtained during theproject funded by Innovate UK (project number 131726) in which the assay was set up (range 26.5 - 70.9 µg/mL).

 

Test substance - RFE

TA2-RFE	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	2000	1000	500	250	125	62.5	31.3	15.6	0.0
% of Negative Control	97.2%	-2.3%	-2.8%	-1.2%	5.3%	44.7%	101.7%	111.0%	93.7%	102.8%
SD	12.0%	0.6%	0.6%	0.5%	6.4%	11.0%	4.4%	7.2%	10.1%	13.0%
% CV	12.31%	-26.95%	-23.01%	-44.65%	122.05%	24.49%	4.29%	6.49%	10.83%	12.66%

Table 2: Cell viability measurements 24 h± 1 h after application of the test substance. NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8.

The calculated IC₅₀ was 119.2 µg/mL.

Main experiments

Three main experiments were performed with a top test substance concentration of 750µg/mL and a dilution factor of 1.5.

A positive control plate was run in parallel, to validate the assay with a top concentration of 100µg/mL and a dilution factor of 1.2.

 

Positive control - ME1

PC-ME1	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	100.0	83.3	69.4	57.9	48.2	40.2	33.5	27.9	0.0
% of Negative Control	107.4%	-1.0%	5.2%	11.5%	13.1%	38.2%	61.6%	77.5%	93.3%	92.6%
SD	3.7%	3.0%	2.4%	13.5%	3.1%	9.8%	4.0%	4.9%	3.3%	17.6%*
% CV	3.43%	-284.96%	47.19%	117.52%	23.57%	25.57%	6.46%	6.38%	3.57%	19.00%

Table 3: Cell viability measurements 24 h ± 1 h after application of positive control (SDS). NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8: *SD value of the NC2 is above 15% (17.6%), therefore one outlier was removed for IC₅₀ calculation. Final results are presented in Table 4.

 

PC-ME1	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	100.0	83.3	69.4	57.9	48.2	40.2	33.5	27.9	0.0
% of Negative Control	104.6%	-1.0%	5.0%	11.2%	12.8%	37.2%	60.0%	75.5%	90.8%	95.4%
SD	3.6%	2.9%	2.4%	13.2%	3.0%	9.5%	3.9%	4.8%	3.2%	12.7%
% CV	3.43%	-284.96%	47.19%	117.52%	23.57%	25.57%	6.46%	6.38%	3.57%	13.30%

Table 4: Cell viability measurements 24 h ± 1 h after application of positive control (SDS). NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8. One outlier was removed for IC₅₀ calculation. Final results are presented here.

The calculated IC₅₀ was 43.71 µg/mL which is the range of the historical data obtained during theproject funded by Innovate UK (project number 131726) in which the assay was set up (range 26.5 - 70.9 µg/mL).

 

Test substance - ME1

TA2-ME1	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	750	500	333	222	148	98.8	65.8	43.9	0.0
% of Negative Control	105.2%	36.4%	18.7%	38.2%	68.4%	57.7%	86.9%	94.0%	92.3%	94.8%
SD	2.8%	7.0%	5.9%	10.1%	11.9%	7.3%	4.5%	7.3%	6.8%	5.9%
% CV	2.70%	19.27%	31.64%	26.38%	17.43%	12.66%	5.14%	7.82%	7.33%	6.27%

Table 5: Cell viability measurements 24 h ± 1 h after application of the test substance. NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8.

The calculated IC₅₀ was 289.9 µg/mL.

 

Positive control - ME2

PC-ME2	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	100.0	83.3	69.4	57.9	48.2	40.2	33.5	27.9	0.0
% of Negative Control	104.9%	19.6%	9.2%	5.1%	28.4%	47.1%	66.0%	78.0%	99.6%	95.1%
SD	7.2%	11.8%	5.7%	14.7%	5.0%	10.0%	4.0%	9.5%	11.0%	9.1%
% CV	6.89%	60.16%	62.39%	290.78%	17.56%	21.24%	6.13%	12.13%	11.07%	9.55%

Table 6: Cell viability measurements 24 h ± 1 h after application of positive control (SDS). NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8.

The calculated IC₅₀ was 47 µg/mL which is in the range of the historical data obtained during the project funded by Innovate UK (project number 131726) in which the assay was set up (range 26.5 - 70.9 µg/mL).

 

Test substance - ME2

TA2-ME2	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	750	500	333	222	148	98.8	65.8	43.9	0.0
% of Negative Control	94.7%	24.3%	26.0%	17.4%	15.5%	32.7%	52.9%	84.3%	117.5%	105.3%
SD	19.2%	11.4%	10.0%	4.5%	12.7%	11.4%	14.8%	8.5%	13.2%	32.1%
% CV	20.32%	46.89%	38.43%	25.85%	81.79%	34.79%	27.97%	10.13%	11.27%	30.51%

Table 7: Cell viability measurements 24 h ± 1 h after application of the test substance. NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8. SD values above 15%, for these conditions, a maximum of 2 outliers were removed for the final calculation. Final results are presented in Table 8.

 

TA2-ME2	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
% of Negative Control	92.9%	22.1%	23.7%	15.9%	14.1%	29.9%	48.3%	76.9%	107.2%	107.1%
SD	8.3%	10.4%	9.1%	4.1%	11.6%	10.4%	13.5%	7.8%	12.1%	13.0%
% CV	8.91%	46.89%	38.43%	25.85%	81.79%	34.79%	27.97%	10.13%	11.27%	12.17%

Table 8: Cell viability measurements 24 h ± 1 h after application of the test substance. NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8. A maximum of 2 outliers were removed. Results of the final calculation are presented here.

The calculated IC₅₀ was 96.8 µg/mL.

 

Positive Control - ME3

PC-ME3	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	100.0	83.3	69.4	57.9	48.2	40.2	33.5	27.9	0.0
% of Negative Control	86.7%	-7.4%	21.0%	-28.1%	-10.8%	3.1%	15.7%	44.7%	101.8%	113.3%
SD	22.9%	12.3%	26.4%	11.4%	18.8%	13.7%	22.3%	27.2%	39.6%	21.4%
% CV	26.4%	-166.6%	126.0%	-40.6%	-174.4%	444.8%	142.0%	60.9%	38.9%	18.9%

Table 9: Cell viability measurements 24 h ± 1 h after application of positive control (SDS). NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8. SD values above 15%, for these conditions, a maximum of 2 outliers were removed for the final calculation. Final results are presented in Table 10.

 

PC-ME3	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	100.0	83.3	69.4	57.9	48.2	40.2	33.5	27.9	0.0
% of Negative Control	88.5%	-6.9%	33.1%	-26.1%	-10.3%	2.9%	25.8%	37.8%	86.4%	111.5%
SD	9.7%	11.5%	16.1%	10.6%	13.3%	12.7%	13.0%	10.0%	12.4%	13.9%
% CV	11.0%	-166.6%	48.6%	-40.6%	-128.7%	444.8%	50.3%	26.5%	14.3%	12.5%

Table 10: Cell viability measurements 24 h ± 1 h after application of positive control (SDS). NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8.

A maximum of 2 outliers were removed. Results of the final calculation are presented here. Note that some values are still above 15%. However, this is not considered to impact the IC₅₀ calculation because calculated value was within historical range.

The calculated IC₅₀ was 32.09 µg/mL which is in the range of the historical data obtained during the project funded by Innovate UK (project number 131726) in which the assay was set up (range 26.5 - 70.9 µg/mL).

  

Test substance - ME3

TA2-ME3	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	750	500	333	222	148	98.8	65.8	43.9	0.0
% of Negative Control	99.3%	1.2%	1.3%	-0.8%	2.4%	18.6%	25.8%	63.7%	79.7%	100.7%
SD	5.6%	1.5%	2.9%	0.9%	3.3%	7.1%	3.3%	9.5%	7.6%	6.7%
% CV	5.59%	126.36%	226.12%	-112.92%	139.17%	38.29%	12.62%	14.95%	9.54%	6.69%

Table 11: Cell viability measurements 24 h ± 1 h after application of the test substance. NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8.

The calculated IC₅₀ was 77.7 µg/mL.

Assay acceptance criteria:

1) Each run includes a Positive Control (SDS) plate with a defined series of concentrations to determine the IC₅₀. In order for the run to be valid, the IC₅₀for SDS must be within the mean ± 1.5 SD of the historical set of runs with this substance (48.7µg/mL ± (1.5x14.8)). - For the RFE, and the 3 ME, the IC₅₀values obtained with the PC were in the range of the historical data obtained during theproject funded by Innovate UK (project number 131726) in which the assay was set up.

2) SD (Standard Deviation) of the 6 values for each condition should be ≤15% (when viability percentage is above 30%) - In some cases, a maximum of 2 outliers was removed to achieve SD ≤15%.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the study conditions, the test substance predicted LD50 was considered to be 300 to 2000 mg/kg bw (Based on in vitro experimental IC50: 77.7 to 289.9 µg/mL).

Executive summary:

An in vitro study was conducted to determine the acute toxicity potential of test substance, 'mono- and di C16-18 PSE and C16-18 AE PSE (10EO)', using cytotoxicity based on Neutral Red Uptake Method, according to OECD Guideline 129, in compliance with GLP. The study was assessed in vitro using XCellR8’s internally validated Human Cell-Based Screen (Non-Regulatory Method). The test uses cultured human dermal fibroblasts in animal product-free culture, Neutral Red Uptake (NRU) method and a prediction model, based on the GHS classification system for acute toxicity. After a 24 h ± 1 h exposure of 8 concentrations of test substance in cell culture medium of Human Dermal Fibroblasts neonatal (HDFn), cytotoxicity was evaluated. Using a prediction model, determined previously, the IC50 value is converted to a corresponding GHS classification for oral acute toxicity. The percentage of viability for each concentration was calculated and normalised to viability results of the negative control (untreated cells) arbitrarily set to 100%. The IC50 (i.e. the concentration at which a decrease in cell viability of 50% was observed) was calculated as being 119.2 µg/mL in the Range Finding Experiment (RFE); 289.9 µg/mL in the main experiment 1 (ME1); 96.8 µg/mL in ME2 and 77.7 µg/mL in ME3. Based on the study results (IC50: 77.7 to 289.9 µg/mL), the study author concluded, the test substance could fall in potential EU CLP category 4 (LD50: 300 to 2000 mg/kg bw) (XCellR8, 2017). However, it is known that the in vitro NRU cytotoxicity assay has a high false positive rate and, therefore, positive results cannot be readily used in a meaningful way in characterising the acutely toxic substances.