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Endpoint summary

Description of key information

Given the high false positive rates of the NRU assay, overall low acute oral toxicity indication from studies on main constituents, the test substance, ‘mono- and di- C16-18 PSE and C16 -18 AE10 PSE’, can be considered to have a low acute oral toxicity potential with LD50 value >2000 mg/kg bw. This is further supported by the low bioavailability potential of the test substance (based on high MW and low water solubility).

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 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

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, 1.5 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.

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).

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.

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%.

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.

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:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Non GLP study

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

no

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

As described in guideline.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Doses:

10 g/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 10 000 mg/kg bw

Based on:

not specified

Mortality:

No deaths occurred.

Clinical signs:

other: The only clinical sign of toxicity was piloerection.

Gross pathology:

No effects.

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Based on the results of the read across study, the LD50 of the test substance, mono- and di- C16-18 PSE + mono- and di- C16-18 PSE, EO-10 is considered to be >10000 mg/kg bw.

Executive summary:

A study was conducted to determine the acute oral toxicity of the read across substance, 'C16 -18 AE1 -2.5' (purity: 100%), in Wistar rats, according to the OECD Guideline 401, standard acute method. The undiluted test substance was administered to groups of five fasted male and female albino rats at a limit dose of 10 gm/kg bw by oral gavage. Animals were observed for 14 d post-dosing. No deaths occurred at the end of the test. No effects were observed in gross pathology and no treatment related changes were observed on body weight. The only clinical sign of toxicity was piloerection. Under the study conditions, the LD50 of the read across substance was determined to be >10000 mg/kg bw (Mϋrmann, 1986). Based on the results of the read across study, a similar LD50 value is considered for the test substance, 'mono- and di C16-18 PSE and C16 -18 AE10 PSE'.

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.

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 >4000 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 >4000 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 >4000 mg a.i./kg bw) (Bremer, 1987). Based on the results of the read across study, a similar oral LD50 value can be considered for the test substance, 'mono- and di- C16 -18 PSE and C16 -18 AE10 PSE'.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Good quality studies

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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 adverse effect observed

Additional information

Oral:

For the acute oral toxicity endpoint, an acute screening study (OECD 129) test is available with the test substance, ‘mono- and di- C16 -18 PSE and C16 -18 AE10 PSE’. Thein vitroNeutral 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 supporting studies available for substances representative of the main constituents, which can be categorised as phosphate esters (PSE), ethoxylated phosphate ester (AE PSE) and free ethoxylated alcohol (AE). As, representative studies are not available for the constituent, AE PSE, the endpoint assessment has been based on representative studies available on PSE and AE only, under the assumption that AE PSE is likely to hydrolyse to AE and PSE. The results are presented below:

Acute screening study with the test substance: An in vitro study was conducted to determine the acute toxicity potential of test substance, ‘mono- and di C16 -18 PSE and C16 -18 AE10 PSE’, using cytotoxicity based on Neutral Red Uptake Method, according to OECD Guideline 129, in compliance with GLP. The study was assessedin vitrousing 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 thein vitroNRU 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 PSE - read across studies:

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) (XXXX, 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 AE - read across study:

A study was conducted to determine the acute oral toxicity of the read across substance, C16 -18 AE (1- 2.5EO) (purity: 100%), in Wistar rats, according to the OECD Guideline 401, standard acute method. The undiluted test substance was administered to groups of five fasted male and female albino rats at a limit dose of 10 gm/kg bw by oral gavage. Animals were observed for 14 d post-dosing. No deaths occurred at the end of the test. No effects were observed in gross pathology and no treatment related changes were observed on body weight. The only clinical sign of toxicity was piloerection. Under the study conditions, the LD50 of the read across substance was determined to be >10000 mg/kg bw (Mϋrmann, 1986).

Further, a HERA 2009 review report on AEs indicated low to moderate order of acute oral toxicity in the rat with LD50 values ranging between 600 to more than 10000 mg/kg bw. The structure of the test compound influenced acute toxicity determined by the relative number of ethoxy units, whereas, carbon chain length was not correlated with the acute oral toxicity. The degree of ethoxylation of the AE appeared to be the only factor found to be of relevance in acute oral toxicity with the compounds with ethoxylate chains between 5 and 14 being more toxic by oral consumption than those with less than 4 or more than 21 ethoxy units. For example, LD50 values for C9-11 AE with 2.5EO ranged from 2.7 to 10 g/kg bw compared to 1.2 to 2.7 g/kg bw for C9-11 AE8. The same trend was observed for C12-14 and C13-15 AE LD50 values; C12-14AE3 (LD50 9.35 g/kg bw) was less acutely toxic than C12-14AE10 (LD50 2.82 g/kg bw), and C13-15AE4 (LD50 > 5g/kg bw) was less acutely toxic than C13-15AE11 (LD50 2.45 g/kg bw). Clinical findings observed in the test animals after treatment were indicative of gastrointestinal irritation such as ulcerations of the stomach, pilo-erection, diarrhoea and lethargy and may be linked with administration of a bolus dose, in particular in cases where the test substance was administered undiluted. The study which resulted in the lowest LD50 value of 600 mg/kg bw for males and 500 mg/kg bw for females were determined for C15-16AE10. It was noted that this study was not in conducted in compliance with OECD guidelines and GLP regulations. C15-16AE10 was administered to four rats of each sex as a 19% w/v solution in water. Diarrhea and lethargy were observed at the highest dose of 1.5 g/kg bw within 24 h. These signs had subsided in surviving animals within 48 h. On the 9th day, two females were underweight; these animals subsequently died on Days 12 and 15. Necropsies at study termination were not conducted.

Overall, given the high false positive rates of the NRU assay, low acute oral toxicity indication from studies on the main constituents, together with the low bioavailability potential of the test substance (based on high MW and low water solubility), the test substance can be considered to have a low acute oral toxicity potential with LD50 value >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. This is also supported by the absence of any systemic effects in the in vivo skin sensitisation studies available for a substance representative of the main constituent, ‘mono- and di- C16 PSE, K+ and H3PO4’ as well as C16-18 AE (1-2.5EO). 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.

Inhalation:

The substance is a solid block 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

Overall based on the available weight of evidence, the test substance, 'mono- and di- C16 -18 PSE and C16 -18 AE10 PSE', is concluded not to warrant classification for acute oral or dermal toxicity, according to the EU CLP criteria (Regulation 1272/2008/EC).