Reaction mass of orthophosphoric acid and potassium dihexadecyl phosphate and dipotassium hexadecyl phosphate

Endpoint summary

Description of key information

Based on the available weight of evidence information from the test substance and read across/direct studies on substances representative of the main constituents, the oral and dermal LD50 values of the test substance, ‘mono- and di- C16 PSE, K+ and H3PO4’, is considered to be >2000 mg/kg bw.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

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

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 end point. 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

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.

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; 1.2 for two Main Experiments (ME1 and ME3); and 1.5 for one Main Experiments (ME2)). The top concentration was previously determined by solubility testing.
Range finding experiment (μg/mL): 200, 100, 50, 25, 12.5, 6.3, 3.1, and 1.6
Main Experiments (ME1 and ME3) (μg/mL): 400, 333.3, 277.8, 231.5, 192.9, 160.8, 134.0, and 111.6 µg/mL
Main Experiment (ME2) (μg/mL): 200, 166.67, 138.89, 115.74, 96.45, 80.38, 66.98 and 55.82

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): Eight different concentration of test substance, with dilution factor of 2 were evaluated: 200, 100, 50, 25, 12.5, 6.3, 3.1, and 1.6 µg/mL
- Two Main Experiments (ME1 and ME3): 400, 333.3, 277.8, 231.5, 192.9, 160.8, 134.0, and 111.6 µg/mL (Dilution Factor 1.2)
- One Main Experiment (ME2): 200, 166.67, 138.89, 115.74, 96.45, 80.38, 66.98 and 55.82 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.

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 0.2 mg/mL (200 µg/mL).

As all viability values obtained with the RFE were above 50%, and further solubility testing showed that the top concentration of 400 µg/mL (0.4 mg/mL) was soluble, two Main Experiments (ME1 and ME3) were performed with a top test substance concentration of 400µg/mL and a dilution factor of 1.2 and one (ME2) with a test substance top concentration of 200 µg/mL (0.2 mg/mL) with a dilution factor of 1.5.

Key result

Dose descriptor:

other: IC50 (Test substance concentration that reduces cell viability to 50% of the negative control)

Remarks:

ME1; ME3

Effect level:

> 400 other: µg/mL

Based on:

test mat.

Remarks on result:

other: As the percentage viability obtained for all concentrations were above 50%, no IC50 was determined. Therefore IC50 value was considered to be greater than highest concentration tested (>400 µg/mL)

Remarks:

Equivalent predicted LD50: 300-2000 mg/kg bw; Potential EU CLP classification: Category 4

Key result

Dose descriptor:

other: IC50 (Test substance concentration that reduces cell viability to 50% of the negative control)

Remarks:

RFE, ME2

Effect level:

> 200 other: μg/mL

Based on:

test mat.

Remarks on result:

other: As the percentage viability obtained for all concentrations were above 50%, no IC50 was determined. Therefore IC50 value was considered to be greater than highest concentration tested (>200 µg/mL

Remarks:

Equivalent predicted LD50: 300-2000 mg/kg bw; Potential EU CLP classification: Category 4

Other findings:

In all assays, no cytotoxicity was observed, even at the top concentration. It was not possible to determine an IC50 value (or IC50 >400 µg/mL) as all percentage of viability obtained were above 50%. Therefore, the test substance was classified as GHS Category 4 “Harmful if swallowed”, suggesting a low acute toxicity potential.

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 - Not soluble

Tier 3: 200 mg/mL in DMSO - Not soluble

Tier 3: 200mg/mL in Ethanol - Not soluble

Tier 4: 0.2mg/mL in cell culture medium - Soluble

Therefore the top concentration used in the range finding experiment (RFE) described here after was 0.2 mg/mL.

As no toxicity was observed in the RFE, further solubility testing was performed to determine a possible higher concentration to use in the Main Experiments (ME). 

Further solubility testing results are: 

200 mg/mL in DMSO - Not soluble

100 mg/mL in DMSO - Not soluble

1.5 mg/mL in cell culture medium - Not soluble

1 mg/mL in cell culture medium - Not soluble

0.8 mg/mL in cell culture medium - Not soluble

0.4 mg/mL in cell culture medium - Soluble

Therefore the concentration of 0.4 mg/mL in cell culture medium was used for ME1 and 3, while 0.2 mg/mL was used for ME2.

Range finding experiment

An initial Range finding experiment (RFE) was performed with a top test substance concentration of 200 μg/mL (0.2 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.

Cell viability measurements 24 h± 1 h after application of test substance / positive control (SDS).

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 and graph. The calculated IC50 was 27.9 µg/mL which is within 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-RFE

TA1-RFE	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	200.0	100.0	50.0	25.0	12.5	6.3	3.1	1.6	0.0
% of Negative Control	95.4%	90.6%	92.0%	90.8%	97.2%	99.3%	94.1%	95.3%	91.3%	104.6%
SD	16.3%	18.0%	14.0%	17.1%	11.3%	14.8%	14.7%	13.0%	12.9%	9.2%
% CV	17.05%	19.84%	15.19%	18.79%	11.64%	14.95%	15.66%	13.64%	14.12%	8.76%

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. A maximum of 2 outliers on the 6 values were removed for the final calculation. Final results are presented in Table 3.

 

 

TA1-RFE	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	200.0	100.0	50.0	25.0	12.5	6.3	3.1	1.6	0.0
% of Negative Control	97.9%	83.9%	89.8%	84.3%	95.0%	96.9%	91.9%	93.0%	89.2%	102.1%
SD	12.3%	15.1%	13.6%	14.4%	11.1%	14.5%	14.4%	12.7%	12.6%	8.9%
% CV	12.55%	17.99%	15.19%	17.04%	11.64%	14.95%	15.66%	13.64%	14.12%	8.76%

Table 3: 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. Note that one value was still above 15%, however,this is not considered to impact the IC₅₀ calculation because all concentrations gave similar results.

As the percentage viability obtained for all concentrations was above 50%, no IC₅₀ was determined.

Main experiments

As all values obtained with the RFE were above 50%, and further solubility testing showed that the top concentration of 400 µg/mL (0.4 mg/mL) was soluble, two Main Experiments (ME1 and ME3) were performed with a top test substance concentration of 400 µg/mL and a dilution factor of 1.2 and one (ME2) with a test substance top concentration of 200 µg/mL (0.2 mg/mL) with a dilution factor of 1.5. A positive control plate was run in parallel, to validate the assay with a top concentration of 100 µg/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 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: *SD value of the NC2 is above 15% (17.6%), therefore one outlier was removed for IC₅₀calculation. Final results are presented in Table 5.

 

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 5: 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 within 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 - ME1 

TA1-ME1	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	400.0	333.3	277.8	231.5	192.9	160.8	134.0	111.6	0.0
% of Negative Control	101.7%	93.9%	88.1%	91.6%	65.7%	88.0%	92.6%	98.6%	92.5%	98.3%
SD	9.8%	7.7%	7.8%	13.1%	22.9%*	11.2%	10.2%	10.1%	9.4%	6.2%
% CV	9.68%	8.24%	8.82%	14.28%	34.81%	12.68%	10.99%	10.21%	10.16%	6.35%

Table 6: 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 on the 6 values were removed for the final calculation. Final results are presented in Table 7.

 

TA1-ME1	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	400.0	333.3	277.8	231.5	192.9	160.8	134.0	111.6	0.0
% of Negative Control	101.7%	93.9%	88.1%	91.6%	56.8%	88.0%	92.6%	98.6%	92.5%	98.3%
SD	9.8%	7.7%	7.8%	13.1%	7.9%	11.2%	10.2%	10.1%	9.4%	6.2%
% CV	9.68%	8.24%	8.82%	14.28%	13.83%	12.68%	10.99%	10.21%	10.16%	6.35%

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. A maximum of 2 outliers were removed. Results of the final calculation are presented here.

 

As the percentage viability obtained for all concentrations was above 50%, no IC₅₀ was determined.

 

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 8: 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 within 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 - ME2

TA1-ME2	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	200.0	166.67	138.89	115.74	96.45	80.38	66.98	55.82	0.0
% of Negative Control	89.6%	120.6%	98.3%	106.7%	98.9%	108.2%	104.1%	115.9%	107.9%	110.4%
SD	18.8%	13.4%	13.4%	4.7%	14.9%	13.1%	13.1%	8.7%	8.0%	11.6%
% CV	20.93%	11.09%	13.59%	4.40%	15.09%	12.08%	12.62%	7.50%	7.41%	10.53%

Table 9: 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. For SD values above 15%, a maximum of 2 outliers were removed for the final calculation. Final results are presented in Table 10.

 

TA1-ME2	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	200.0	166.67	138.89	115.74	96.45	80.38	66.98	55.82	0.0
% of Negative Control	82.9%	128.0%	104.4%	113.2%	104.9%	114.8%	110.4%	122.9%	114.5%	117.1%
SD	7.9%	14.2%	14.2%	5.0%	15.8%	13.9%	13.9%	9.2%	8.5%	12.3%
% CV	9.56%	11.09%	13.59%	4.40%	15.09%	12.08%	12.62%	7.50%	7.41%	10.53%

Table 10: 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.

 

As the percentage viability obtained for all concentrations was above 50%, no IC₅₀ was determined.

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 11: Cell viability measurements 24h± 1h after application of positive control (SDS). NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8. For SD values above 15%, a maximum of 2 outliers were removed for the final calculation. Final results are presented in Table 12.

 

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 12: Cell viability measurements 24h± 1h 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 within 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

TA1-ME3	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	400.0	333.3	277.8	231.5	192.9	160.8	134.0	111.6	0.0
% of Negative Control	92.9%	95.2%	92.4%	97.1%	94.7%	99.4%	97.5%	97.9%	99.6%	107.1%
SD	12.9%	3.5%	8.6%	5.9%	14.2%	11.5%	6.0%	9.3%	18.9%	13.2%
% CV	13.83%	3.72%	9.33%	6.10%	15.00%	11.55%	6.11%	9.53%	19.00%	12.30%

Table 13: 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. For SD values above 15%, a maximum of 2 outliers on the 6 values were removed for the final calculation. Final results are presented in Table 14.

 

TA1-ME3	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	400.0	333.3	277.8	231.5	192.9	160.8	134.0	111.6	0.0
% of Negative Control	92.9%	95.2%	92.4%	97.1%	94.7%	99.4%	97.5%	97.9%	105.9%	107.1%
SD	12.9%	3.5%	8.6%	5.9%	14.2%	11.5%	6.0%	9.3%	12.1%	13.2%
% CV	13.83%	3.72%	9.33%	6.10%	15.00%	11.55%	6.11%	9.53%	11.43%	12.30%

Table 14: 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 when SD values above 15%. Results of the final calculation are presented here.

 

As the percentage viability obtained for all concentrations was above 50%, no IC₅₀ was determined.

Assay acceptance criteria

Results were checked against the following 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 IC₅₀ for SDS must be within the mean ± 1.5 SD of the historical set of runs with this substance (48.7 µg/mL ± (1.5 x 14.8)). 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%.

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: >400 μg/mL).

Executive summary:

An in vitro study was conducted to determine the acute toxicity potential of test substance, 'mono- and di- C16 PSE, K+ and H3PO4', 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 was converted to a corresponding GHS classification for oral acute toxicity. The solubility was first performed to determine the top concentration for the Range Finding Experiment (RFE). 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 >200 μg/mL in the RFE. As no toxicity was observed in the RFE, further solubility testing was performed to determine a possible higher concentration to use in the Main Experiments (ME). Therefore the highest concentration of 400 μg/mL in cell culture medium was used for ME1 and 3, while 200 μg/mL was used for ME2. In all test substance assays, no cytotoxicity was observed, even at the top concentration. It was not possible to determine an IC50 value as all percentage of viability obtained were above 50%. Based on the study results (IC50: >400 μg/mL), the study author concluded, the test substance could fall in potential EU CLP category 4 (LD50: 300 to 2000 mg/ kg bw).

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From August 30, 1985 to September 13, 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: notification No. 118 of the Pharmaceuticals Affairs Bureau, 15 Feb 1984, Toxicity Test Guideline

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

not specified

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

other: CFY (Sprague-Dawley origin)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
- Source: Interfauna UK Limited, Huntingdon, Cambridgeshire, England
- Age at study initiation: 4 to 6 weeks
- Weight at study initiation: 109 to 150 g
- Fasting period before study: yes; overnight prior to and 4 h after dosing
- Housing: in groups by sex in metal cages with wire mesh floor
- Diet (e.g. ad libitum): standard laboratory rodent diet (Labsure LAD 1), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum 8 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23°C
- Humidity (%): 62% mean
- Air changes (per hr): ca. 15/h
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on oral exposure:

Vehicle
- Concentration in vehicle: 80%
- Amount of vehicle (if gavage): 20 mL/kg bw

Doses:

0, 16.0 g/kg bw

No. of animals per sex per dose:

preliminary study: 2
main study: 10

Control animals:

yes

Details on study design:

- Duration of observation period following administration: preliminary study 5 d; main study 14 d
- Frequency of observations and weighing: (a) bodyweights: Day 1 (day of dosing), 4, 8, 15 (b) clinical signs: soon after dosing, then at frequent intervals for the remainder of Day 1. On subsequent days the animals were observed at least once in the morning and once at the end of the experimental day (on Saturdays and Sundays app. 11:30 a.m.)
- Necropsy of survivors performed: yes

Statistics:

none

Preliminary study:

0/4 animals died in the preliminary test.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 16 000 mg/kg bw

Based on:

test mat.

Mortality:

0/20 animals died in the main study

Clinical signs:

other: - Piloerection in 20/20 animals in treated group; recovery on Day 3 - No clinical signs in control group

Gross pathology:

- Terminal autopsy findings were normal

Other findings:

None

Interpretation of results:

other: Not classified based on EU CLP criteria

Conclusions:

Under the study conditions, the oral LD50 in rats for test substance was determined to be >16000 mg/kg bw.

Executive summary:

A study was conducted to determine the acute toxicity of the read across substance, di- C16 PSE (purity: 100%), according to the notification no. 118 of the Pharmaceuticals Affairs Bureau, 15 Feb 1984, Toxicity Test Guideline (similar to OECD guideline 401). Groups of fasted, 4 to 6 weeks old CFY (Sprague-Dawley origin) rats, 10/sex were given a single oral dose of test substance in distilled water at doses of 0 (control) and 16 g/kg bw and observed for 14 d. No mortality occurred. Piloerection was observed in all animals in the treated group, however, the animals had recovered on Day 3. No effects on body weight were observed. Terminal necropsy findings were found to be normal. Under the study conditions, the oral LD50 in rats for test substance was determined to be >16000 mg/kg bw (Huntingdon, 1985). Based on the results of the read across study, similar absence of toxicity and oral LD50 value can be expected for the test substance, 'mono- and di- C16 PSE, K+ and H3PO4'.

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From June 02, 1987 to June 17, 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: Ibm: RORO (SPF), also known as Fü-albino SPF rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
- Age at study initiation: about 6 weeks
- Weight at study initiation: female 114-117 g; male 116-124 g
- Fasting period before study: 18 h
- Housing:
- Diet: NAFAG standard rat maintenance diet, No. 850 (cubic), ad libitum
- Water: tap water, ad libitum
- Acclimatisation period: seven days under laboratory conditions

Environmental conditions
- Temperature: 20-24°C
- Humidity: 45-65 %
- Air changes: air-conditioned room
- Photoperiod: 12/12 h dark / light

Route of administration:

oral: gavage

Vehicle:

other: Standard Suspending Vehicle (SSV), please see below in " Details on oral exposure"

Details on oral exposure:

Vehicle
The test article was suspended in Standard Suspending Vehicle (SSV)
1000 mL SSV contain:
5 g sodium carboxy methyl cellulose of median viscosity,
4 mL Tween 80,
5 mL benzylalcohol pro analysis,
9 g sodium-chloride pro analysis,
aqua destillata ad 1000 mL.

- Amount of vehicle: 10 mL/kg bw

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5 animals per sex

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 d
- Frequency of observations and weighing: daily for clinical signs and weighing on Day 1, 4, 8, 11, 15
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs (respiratory distress, crust around nose, hunched posture, crust around eyes, exitability), body weight, histopathology

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: i.e., equivalent to >4250 mg a.i./kg bw

Mortality:

No compound-related deaths occurred.
One male was found dead early in the morning of Day 8. This case of death was considered to be a result of an application injury. This male rat showed a marked respiratory distress and a marked loss of weight. The histopathological examination of the lung of this animal revealed aspiration pneumonia.

Clinical signs:

other: The main symptom was respiratory distress seen in 3 males and 1 female. This symptom developed in consequence of aspiration of a little test suspension. The other findings were of no toxicological significance.

Gross pathology:

In the urinary bladder of male rat 2694, gritty contents were observed. This finding was of a spontaneous nature. No other macroscopic organ changes were seen.

Other findings:

Histopathology: Bronchopneumonia caused the respiratory distress and itself was caused by aspiration of test suspension (by the male rat that died at Day 8).

Interpretation of results:

other: not classified based on EU CLP Criteria

Conclusions:

Based on the results of the read across study, LD50 for the test substance, is considered to be >4250 mg/kg bw.

Executive summary:

A study was conducted to determine the acute oral toxicity of the read across substance, mono- and di- C16 PSE, K+ (purity: ca. 85%), according to the OECD Guideline 401, standard acute method, in compliance with GLP. Five male and 5 female Fü-albino SPF rats were randomly selected for an acute oral toxicity study. Fasted rats were given a single dose of the test substance suspended in SSV (Standard Suspended Vehicle) by gavage at a dose level of 5000 mg/kg bw. They were observed for 15 d for toxic signs, mortality and body weight changes. All rats were examined for gross lesions. No compound-related deaths occurred. No compound-related incompatibility reactions were observed. No compound-related effect on body weight development appeared. No compound-related gross or microscopic lesions were observed. The LD50 was determined at >5000 mg/kg bw (i.e., equivalent to >4250 mg a.i./kg bw) (XXXX, 1987). Based on the results of the read across study, similar absence of toxicity and oral LD50 value can be expected for the test substance, 'mono- and di- C16 PSE, K+ and H3PO4'.

Reference 4

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1975-1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

A study was conducted to determine the acute toxicity in rats of test substance, phosphoric acid (75 -85% aqueous solution) in Sprague Dawley rats.

GLP compliance:

not specified

Remarks:

The original study was from the pre-GLP period.

Test type:

other: Calculation of LD50s was done according to the method of E.J. de Beer (1945; Journal of Pharmacology and Experimental Therapeutics, 85:1)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Mix of male and female Sprague-Dawley albiono rats

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Doses:

2,510, 3,160, 3,980, 5,010 and 6,310 mg/kg bw (5,010 mg/kg bw was the highest test dose for PA 85 %)

No. of animals per sex per dose:

5

Control animals:

not specified

Details on study design:

A 14-d observation period followed administration.
Calculation of LD50s was done according to the method of E.J. de Beer (1945; Journal of Pharmacology and Experimental Therapeutics, 85:1)

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

ca. 3 500 mg/kg bw

Based on:

test mat.

Remarks:

85% test substance

95% CL:

>= 3 150 - <= 3 890

Remarks on result:

other: equivalent to 2975 mg a.i./kg bw

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

ca. 4 200 mg/kg bw

Based on:

test mat.

Remarks:

80% test substance

95% CL:

>= 3 780 - <= 4 660

Remarks on result:

other: equivalent to 3360 mg a.i./kg bw

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

ca. 4 400 mg/kg bw

Based on:

test mat.

Remarks:

75% test substance

95% CL:

>= 3 870 - <= 4 970

Remarks on result:

other: equivalent to 3300 mg a.i./kg bw

Key result

Sex:

male/female

Dose descriptor:

LD0

Effect level:

ca. 2 510 mg/kg bw

Based on:

test mat.

Remarks:

85% test substance

Remarks on result:

other: equivalent to 2133.5 mg a.i./kg bw

Key result

Sex:

male/female

Dose descriptor:

LD0

Effect level:

ca. 3 160 mg/kg bw

Based on:

test mat.

Remarks:

80% test substance

Remarks on result:

other: equivalent to 2528 mg a.i./kg bw

Key result

Sex:

male/female

Dose descriptor:

LD0

Effect level:

ca. 3 160 mg/kg bw

Based on:

test mat.

Remarks:

75% test substance

Remarks on result:

other: equivalent to 2370 mg a.i./kg bw

Mortality:

One male of 2510 mg/kg bw treated group, 1 female of 3160 mg/kg bw treated group, 1 male and 3 female of 3980 mg/kg bw treated group and 3 male and 2 female of 5010 mg/kg bw treated group rats were died after one to two days administration.

Clinical signs:

other: Reduced appetite and activity (one to three days in survivors), increasing weakness, collapse and death.

Gross pathology:

Hemorrhagic areas of the lungs, liver hyperemia, and hemorrhaging and necrosis of gastrointestinal tract.

Other findings:

Survivors (14 d): Viscera appeared normal.

Results:

Average initial weight and mortality (85% phosphoric acid, undiluted)

Dosage (mg/kg)	Average initial weight		Mortalities/Dosed			Time of mortality
	Male	Female	Male	Female	Combined	One to two days
2510	225	225	1/2	0/3	1/5
3160	225	240	0/3	1/2	1/5
3980	225	230	1/2	3/3	4/5
5010	220	215	3/3	2/2	5/5

The calculated oral LD50s for PA 85 %, PA 80 % and PA 75 % were 3,500, 4,200 and 4,400 mg test material/kg, respectively (95% confidence limits 3,150-3,890, 3,780-4,660 and 3,870-4,970 mg/kg); LDLo were 2,510, 3,160 and 3,160 mg/kg, respectively.

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Under the study conditions, the test substance LD50 in rats were determined to range from 2975-3360 mg a.i./kg bw.

Executive summary:

A study was conducted to determine the acute toxicity in rats of test substance, phosphoric acid (75 -85% aqueous solution) in Sprague Dawley rats. The test substancewas administered by gavages to groups of 5 (mix of male and female) Sprague-Dawley albino rats at dose of 2,510, 3,160, 5,010 and 6,310 mg/kg bw. The animals were observed for 14 d following administration for mortality and other signs of toxicities. Calculation of LD50 was performed according method described by Beer (1945). The calculated LD50 values were determined to be 3500 mg/kg bw (2975 mg a.i./kg bw), 4200 mg/kg bw (3360 mg a.i./kg bw) and 4400 mg/kg bw (3300 mg a.i./kg bw) for 85%, 80% and 75% test substance, respectively (OECD SIDS, 2009).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Guideline compliant studies or studies meeting standard methods

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For the acute oral toxicity endpoint, an acute screening study (OECD 129) test is available with the test substance, ‘mono- and di- C16 PSE, K+ and H3PO4'. Thein vitro Neutral Red Uptake (NRU) cytotoxicity screening study according to the ECHA R.7a Guidance, cannot be used as a stand-alone test, but could be used within a WoE approach to adapt the standard information requirements for acute oral toxicity. Therefore, the acute oral toxicity endpoint assessment has been based on the acute oral screening study available on the test substance along with oral toxicity studies on substances representative of the main constituents, which can be categorised as phosphate esters (PSE) and phosphoric acid (H3PO4). The results are presented below:

NRU acute toxicity screening study with test substance:

An in vitro study was conducted to determine the acute toxicity potential of test substance, 'mono- and di- C16 PSE, K+ and H3PO4',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 was converted to a corresponding GHS classification for oral acute toxicity. The solubility was first performed to determine the top concentration for the Range Finding Experiment (RFE). 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 >200 μg/mL in the RFE. As no toxicity was observed in the RFE, further solubility testing was performed to determine a possible higher concentration to use in the Main Experiments (ME). Therefore the highest concentration of 400 μg/mL in cell culture medium was used for ME1 and 3, while 200 μg/mL was used for ME2. In all test substance assays, no cytotoxicity was observed, even at the top concentration. It was not possible to determine an IC50 value as all percentage of viability obtained were above 50%. Based on the study results (IC50: >400 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.

Constituent 1: PSE

Study 1: A study was conducted to determine the acute oral toxicity of the read across substance, mono- and di- C16 PSE, K+ (purity: ca. 85%), according to the OECD Guideline 401, standard acute method, in compliance with GLP. Five male and 5 female Fü-albino SPF rats were randomly selected for an acute oral toxicity study. Fasted rats were given a single dose of the test substance suspended in SSV (Standard Suspended Vehicle) by gavage at a dose level of 5000 mg/kg bw. They were observed for 15 d for toxic signs, mortality and body weight changes. All rats were examined for gross lesions. No compound-related deaths occurred. No compound-related incompatibility reactions were observed. No compound-related effect on body weight development appeared. No compound-related gross or microscopic lesions were observed. The LD50 was determined at >5000 mg/kg bw (i.e., equivalent to >4250 mg a.i./kg bw) (Bremer, 1987).

Study 2: A study was conducted to determine the acute toxicity of the read across substance, di- C16 PSE (purity: 100%), according to the notification no. 118 of the Pharmaceuticals Affairs Bureau, 15 Feb 1984, Toxicity Test Guideline (similar to OECD guideline 401). Groups of fasted, 4 to 6 weeks old CFY (Sprague-Dawley origin) rats, 10/sex were given a single oral dose of test substance in distilled water at doses of 0 (control) and 16 g/kg bw and observed for 14 d. No mortality occurred. Piloerection was observed in all animals in the treated group, however, the animals had recovered on Day 3. No effects on body weight were observed. Terminal necropsy findings were found to be normal. Under the study conditions, the oral LD50 in rats for test substance was determined to be >16000 mg/kg bw (Huntingdon, 1985).

Constituent 2: phosphoric acid

A study was conducted to determine the acute toxicity in rats of test substance, phosphoric acid (75 -85% aqueous solution) in Sprague Dawley rats. The test substancewas administered by gavages to groups of 5 (mix of male and female) Sprague-Dawley albino rats at dose of 2,510, 3,160, 5,010 and 6,310 mg/kg bw. The animals were observed for 14 d following administration for mortality and other signs of toxicities. Calculation of LD50 was performed according method described by Beer (1945). The calculated LD50 values were determined to be 3500 mg/kg bw (2975 mg a.i./kg bw), 4200 mg/kg bw (3360 mg a.i./kg bw) and 4400 mg/kg bw (3300 mg a.i./kg bw) for 85%, 80% and 75% test substance, respectively (OECD SIDS, 2009).

Overall, the test substance can be considered to have a low acute oral toxicity, with LD50 values for its main constituents >2000 mg/kg bw.

Dermal:

As per Annex VIII (8.5.3), the acute dermal toxicity testing is not needed as the substance does not meet the criteria for classification for acute toxicity and STOT SE for the oral route (based on read across studies). This is also supported by the absence of any systemic effects in thein vivoskin sensitisation study available with the test substance, ‘mono- and di- C16 PSE, K+ and H3PO4’. Moreover, given the physico-chemical properties of the test substance, the dermal LD50 value is less likely (due to lower absorption potential of dermal route) to be lower than oral LD50 or the oral doses showing clinical signs. Hence, testing via dermal route will less likely result in any additional hazard identification and testing is therefore considered unnecessary. No systemic toxicity was observed in thein vivoskin sensitisation study available with the read across substance.

Inhalation:

The substance is a solid powder (particle size exceeding 2000 µm) with a low vapour pressure at room temperature. Due to its physical state and physical chemical properties it is unlikely that this substance will form inhalable dust, mist or fumes during normal processing and use conditions. In case inhalable forms of the substance are created under particular conditions (e. g. spraying, elevated temperature/pressure), appropriate risk management measures such as closed systems, exhaust ventilation or wearing of respirators are implemented to control exposure. Under such conditions, the risk to humans following inhalation exposure can be considered minimal and further testing involving vertebrate animals may be omitted, in accordance with Annex XI (1.2) of the REACH regulation.

Justification for classification or non-classification

Based on the available weight of evidence information from the test substance and read across/direct studies with the main constituents, the test substance, 'mono- and di- C16 PSE, K+ and H3PO4', does not warrant classification for acute oral or dermal toxicity, according to the EU CLP (Regulation 1272/2008/EC).