Potassium Lauroyl Wheat Amino Acids

Endpoint summary

Description of key information

Overall, given the high false positive rates of the NRU assay, low acute oral toxicity indication from studies on the main constituents together with innocuousness of hydrolysed proteins/amino acids, the test substance can be considered to have a low acute oral toxicity potential with LD50 value exceeding 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 29, 2017 to September 06, 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; 1.5 for 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 Experiment (μg/mL): 500, 333.3, 222.2, 148.1, 98.8, 65.8, 43.9, 29.3

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 (μg/mL): 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6
Main Experiment (μg/mL): 500, 333.3, 222.2, 148.1, 98.8, 65.8, 43.9, 29.3

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 2 mg/mL (2000 µg/mL).

Key result

Dose descriptor:

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

Remarks:

Test substance (3 Experiments range)

Effect level:

ca. 129.5 - ca. 147.4 other: µg/mL

Based on:

test mat.

Remarks on result:

other:

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:

Positive control (3 Experiments range)

Effect level:

ca. 37.8 - ca. 45.4 other: μg/mL

Based on:

test mat.

Remarks on result:

other:

Remarks:

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

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

Range Finding Experiment

An initial 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 100 µg/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	102.5%	-0.9%	-0.6%	10.4%	51.6%	60.4%	81.5%	97.9%	108.3%	97.5%
SD	6.9%	0.9%	0.9%	3.5%	7.2%	10.1%	5.0%	4.8%	5.1%	2.1%
% CV	6.73%	-100.78%	-145.76%	33.83%	14.01%	16.65%	6.12%	4.94%	4.75%	2.15%

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.

 

The calculated IC₅₀ was 58.3 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	2000	1000	500	250	125	62.5	31.3	15.6	0.0
% of Negative Control	107.5%	-0.7%	-0.8%	0.3%	-0.9%	52.3%	94.6%	93.9%	93.4%	92.5%
SD	5.4%	1.1%	0.9%	1.4%	1.4%	5.1%	3.4%	2.0%	3.9%	3.5%
% CV	5.04%	-143.00%	-107.01%	514.05%	-160.96%	9.79%	3.56%	2.12%	4.15%	3.84%

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 IC₅₀was 130.46 µg/mL.

Main Experiments

Three Main Experiments were performed, with a top test substance concentration of 500 µg/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 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	86.5%	5.4%	1.9%	9.0%	25.6%	23.9%	54.8%	83.8%	113.2%	113.5%
SD	22.2%	7.3%	8.3%	4.8%	6.9%	4.8%	13.0%	12.7%	16.0%	25.0%
% CV	25.63%	134.65%	429.11%	53.26%	27.09%	20.02%	23.81%	15.13%	14.11%	22.01%

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: *For SD value above 15%, a maximum of 2 outliers were removed for the final 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	99.7%	5.5%	2.0%	9.2%	26.0%	24.3%	55.6%	85.0%	121.2%	100.3%
SD	16.4%	7.4%	8.4%	4.9%	7.0%	4.9%	13.2%	12.9%	5.6%	12.7%
% CV	16.41%	134.65%	429.11%	53.26%	27.09%	20.02%	23.81%	15.13%	4.60%	12.62%

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. A maximum of 2 outliers were removed from calculation was removed for IC₅₀ calculation. Final results are presented here. Note that SD value for the NC1 was still above 15%, however, this is not considered to impact the IC₅₀ calculation because calculated value was within historical range.

 

The calculated IC₅₀ was 45.4 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	500.0	333.3	222.2	148.1	98.8	65.8	43.9	29.3	0.0
% of Negative Control	82.4%	0.4%	-3.1%	3.3%	49.5%	86.3%	107.3%	118.0%	119.2%	117.6%
SD	17.4%	2.9%	4.7%	2.3%	8.0%	8.2%	5.8%	5.6%	10.7%	14.6%
% CV	21.10%	769.70%	-152.28%	69.37%	16.11%	9.55%	5.36%	4.71%	8.95%	12.45%

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 IC₅₀was 147.4 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	84.2%	1.5%	3.5%	2.3%	14.9%	23.3%	44.6%	72.4%	115.8%	115.8%
SD	29.5%	3.0%	5.8%	1.4%	6.9%	6.9%	4.6%	7.0%	5.8%	15.9%
% CV	34.99%	204.55%	163.63%	62.45%	46.50%	29.74%	10.26%	9.74%	5.02%	13.77%

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. *For SD value above 15%, a maximum of 2 outliers were removed for the final calculation. Final results are presented in Table 7.

 

 

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	89.8%	1.4%	3.2%	2.1%	13.6%	21.4%	40.8%	66.2%	105.9%	110.2%
SD	23.7%	2.8%	5.3%	1.3%	6.3%	6.4%	4.2%	6.4%	5.3%	11.3%
% CV	26.42%	204.55%	163.63%	62.45%	46.50%	29.74%	10.26%	9.74%	5.02%	10.25%

Table 7: 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. maximum of 2 outliers were removed from calculation was removed for IC₅₀calculation. Final results are presented here. Note that SD value for the NC1 was still above 15%. However, this is not considered to impact the IC₅₀ calculation because calculated value was within historical range.

 

The calculated IC₅₀ was 37.8 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

TA1-ME2	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	500.0	333.3	222.2	148.1	98.8	65.8	43.9	29.3	0.0
% of Negative Control	97.6%	2.0%	5.9%	6.7%	42.8%	61.8%	87.2%	96.7%	107.7%	102.4%
SD	7.7%	7.0%	12.6%	4.8%	5.6%	5.7%	5.4%	9.3%	7.2%	13.1%
% CV	7.86%	355.03%	214.31%	71.40%	13.10%	9.18%	6.24%	9.57%	6.69%	12.81%

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.

The calculated IC₅₀ was 129.5 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	102.0%	-3.5%	-3.5%	12.6%	18.8%	35.8%	58.2%	85.9%	117.2%	98.0%
SD	9.2%	9.5%	12.8%	11.9%	6.7%	8.6%	5.6%	6.5%	5.0%	5.2%
% CV	9.01%	-273.90%	-369.22%	94.87%	35.65%	23.95%	9.64%	7.53%	4.26%	5.28%

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.

The calculated IC₅₀ was 43.1 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

TA1-ME3	NC1	C1	C2	C3	C4	C5	C6	C7	C8	NC2
Concentration (µg/mL)	0.0	500.0	333.3	222.2	148.1	98.8	65.8	43.9	29.3	0.0
% of Negative Control	101.3%	2.8%	-1.2%	24.6%	44.4%	70.0%	94.6%	99.0%	113.0%	98.7%
SD	11.6%	7.1%	9.3%	3.8%	4.7%	5.0%	7.9%	9.7%	11.0%	3.7%
% CV	11.41%	254.34%	-755.10%	15.37%	10.49%	7.16%	8.36%	9.81%	9.76%	3.75%

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.

 

The calculated IC₅₀ was 137.4 µg/mL.

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 [48.7μg/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%.

 

Acute toxicity Prediction Model and Interpretation of the Results

 

During the internal validation of the assay, using 20 cosmetic ingredients generating an in vitro IC50 value (IC50= the time taken to reduce cell viability to 50% of the negative control), a prediction model was used to convert the IC50 value to a corresponding GHS classification for oral acute toxicity, with an accuracy of 66.7% (at least equivalent to the accuracy of the traditional animal-based LD50 test).

Prediction model for in vitro acute toxicity determination of IC50 values, determined during internal validation of the assay:

	Fatal if Swallowed	Toxic if Swallowed	Harmful if Swallowed	Potentially harmful if Swallowed
LD50	5-50 mg/kg	50-300 mg/kg bw	300-2000 mg/kg bw	2000-5000 mg/kg bw
GHS	Categories 1-2	Category 3	Category 4	Category 5
IC50	ND	< 10 µg/mL	10-1000 µg/mL	>1000 µg/mL

In the present study, 8 concentrations of the test substance were applied to HDFn cells in culture medium for 24 h ±1 h and the IC₅₀ was calculated. The table below shows the IC₅₀ values as well as the potential corresponding GHS (Global Harmonized System) classification. 

 

	Calculated IC50	GHS classification
RFE	130.5µg/mL	Category 4
ME1	147.4 µg/mL	Category 4
ME2	129.5 µg/mL	Category 4
ME3	137.4 µg/mL	Category 4

 

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

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: 129.5 to 147.4 μg/mL).

Executive summary:

An in vitro study was conducted to determine the acute toxicity potential of test substance, 'potassium lauroyl wheat amino acids' (active: 69.2%), using cytotoxicity based Neutral Red Uptake (NRU) 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, 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 (500, 333.3, 222.2, 148.1, 98.8, 65.8, 43.9, 29.3 μg/mL) and positive control (100, 83.3, 69.4, 57.9, 48.2, 40.2, 33.5, 27.9 μg/mL Sodium dodecyl sulphate) 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. 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 130.46 μg/mL in the range finding experiment. The IC50 value obtained in all three main experiments were between 10-1000 µg/mL (147.4, 129.5 and 137.4 μg/mL). Based on the study results (IC50: 129.5 to 147.4 μg/mL), the study author concluded, the test substance could fall in potential EU CLP category 4 (LD50: 300 to 2000 mg/ kg bw). For the range finding and the main experiments, the IC50 values obtained with the positive control were in the range of the historical data obtained. In some cases, a maximum of 2 outliers was removed to achieve standard deviation (SD) ≤15%. Under the study conditions, the predicted LD50 value of the test substance was considered to lie between 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:

From October 25, 1989 to November 08, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals
- Source: Bantin & Kingman Ltd., Grimston, Aldborough, Hull, U.K.
- Age at study initiation: 5-8 weeks
- Weight at study initiation: male: 120-145g; female: 120-137g
- Fasting period before study: overnight immediately before dosing and for approximately two hours after dosing
- Housing: in groups of 5 by sex in polypropylene cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 d

Environmental conditions
- Temperature (°C): 20-24
- Humidity (%): 51-66
- Air changes (per h): 15
- Photoperiod (h dark / h light): 12/12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Maximum dose volume: 1.94 mL/kg bw

Doses:

Single dose of 2000 mg/kg bw

No. of animals per sex per dose:

10 (5 males and 5 females)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 d
- Frequency of observations and weighing: once a day; bw were recorded on Day 0, 7 and 14
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weights, histopathology

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

no mortality occured.

Clinical signs:

other: No evidence of systemic toxicity was noted during the study period.

Gross pathology:

No abnormalities were noted at necropsy of animals killed at the end of the study.

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Based on the results of the read across study, the test substance, Potassium lauroyl wheat amino acids LD50 is considered to be >2000 mg/kg bw.

Executive summary:

A study was conducted to determine the acute toxicity potential of the read across substance, 'sodium cocoyl glutamate' in rats using standard acute method, according to OECD Guideline 401, in compliance with GLP. A group of 10 fasted Sprague-Dawley Rats (five males and five females) were given a single oral dose of undiluted test material at a dose level of 2000 mg/kg bw (limit test). Animals were observed up to 14 d for signs of toxicity and mortality. There were no deaths recorded during the observation period. No toxicologically significant effects on bodyweight or any other evidence of systemic toxicity were noted during the study period. No abnormalities were noted at necropsy of animals killed at the end of the study. Under the study conditions, oral LD50 value of the read across substance was determined to be >2000 mg/kg bw (Safepharm, 1989). Based on the results of the read across study, similar oral LD50 can be expected for the test substance, 'potassium lauroyl wheat amino acids'.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Guideline compliant study

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

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, ‘potassium lauroyl wheat amino acids’. The in 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, together with oral toxicity study on substance representative of the main constituent and general weight of evidence from hydrolysed proteins (to address the toxicity impact of the different combinations of amino acid alkyl amides). The results are presented below:

Study 1:An in vitro study was conducted to determine the acute toxicity potential of test substance, 'potassium lauroyl wheat amino acids' (active: 69.2%), using cytotoxicity based Neutral Red Uptake (NRU) 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, 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 (500, 333.3, 222.2, 148.1, 98.8, 65.8, 43.9, 29.3 μg/mL) and positive control (100, 83.3, 69.4, 57.9, 48.2, 40.2, 33.5, 27.9 μg/mL Sodium dodecyl sulphate) 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. 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 130.46 μg/mL in the range finding experiment. The IC50 value obtained in all three main experiments were between 10-1000 µg/mL (147.4, 129.5 and 137.4 μg/mL). Based on the study results (IC50: 129.5 to 147.4 μg/mL), the study author concluded, the test substance could fall in potential EU CLP category 4 (LD50: 300 to 2000 mg/ kg bw). For the range finding and the main experiments, the IC50 values obtained with the positive control were in the range of the historical data obtained. In some cases, a maximum of 2 outliers was removed to achieve standard deviation (SD) ≤15%. Under the study conditions, the predicted LD50 value of the test substance was considered to lie between 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.

 

Study 2: A study was conducted to determine the acute toxicity potential of the read across substance, 'sodium cocoyl glutamate' in rats using standard acute method, according to OECD Guideline 401, in compliance with GLP. A group of 10 fasted Sprague-Dawley Rats (five males and five females) were given a single oral dose of undiluted test material at a dose level of 2000 mg/kg bw (limit test). Animals were observed up to 14 d for signs of toxicity and mortality. There were no deaths recorded during the observation period. No toxicologically significant effects on bodyweight or any other evidence of systemic toxicity were noted during the study period. No abnormalities were noted at necropsy of animals killed at the end of the study. Under the study conditions, oral LD50 value of the read across substance was determined to be >2000 mg/kg bw (Safepharm, 1989).Based on the results of the read across study, similar oral LD50 can be expected for the test substance, 'potassium lauroyl wheat amino acids'.

The absence of toxicity in the above study is further supported by the evidence that the different amino acid combinations with alky amide are not likely have an impact on the endpoint. This conclusion is based on the below facts:

-      Hydrolysed proteins are natural compounds derived by the hydrolysis of tissues from living organisms that can be of plant or animal origin. The animal and vegetable cells are formed mainly by proteins, which constitute more than the half of the dry weight of the cell. Proteins determine the shape and structure of the cell and also function as an instrument of molecular recognition and of catalysis (Alberts Bruce, 1986).

-      Proteins have many different biological functions. The widest group of proteins are the enzymes whose function is about catalysing the biochemical processes that take place in the living organisms. Moreover, there are proteins of reservation of amino acids such as plant nutrients; transport proteins of specific molecules; proteins that work as essential elements of the motile and contractile systems; protective proteins that are present in the blood of the vertebrates such as antibodies; proteins that function as hormones and, finally, structural proteins (Lehninger, 1983).

-      The proteins that are found in food and eaten by human beings and mammals are normally degraded metabolically by means of enzymatic processes to give rise to more simple metabolites (peptides and amino acids) that are used by the live cells for the biosynthesis of new specific proteins. The hydrolysed proteins coming from the enzymatic hydrolysis of the animal tissues, therefore, do not cause any danger to human beings and mammals in general. Proteins appear in all biochemical processes that take place in every live cell being, this way they are considered as essential compounds for human life.

-      The hydrolysed proteins are authorized by the EU in order to be used as attractant in the elaboration of baits in combination with appropriate insecticides of the Organic Farming (Regulation EC 1488/97 annex 2, part B). This shows the innocuousness of these compounds, since the practice of this kind of agriculture is very demanding with the use of products that can be harmful to human beings. Also in Regulation (EC) No 1774/2002 of the European Parliament and the Council of 3 October 2002 laying down health rules concerning animal by-products not intended for human consumption, hydrolysed proteins (molecular weight <10,000 Dalton) have been allowed in feed animal products, ensuring the safety and the toxicology harmlessness of them.

Overall, given the high false positive rates of the NRU assay, low acute oral toxicity indication from studies on the main constituents together with innocuousness of hydrolysed proteins/amino acids, the test substance can be considered to have a low acute oral toxicity potential with LD50 value >2000 mg/kg bw.

References:

1) Molecular biology of the cell, by Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith Roberts, and James Watson; Garland Publ. Inc., New York, 1146 pp, January, 1986.

(2) Principles of biochemistry by Albert L Lehninger. pp 1011. Worth Publishers, New York, January, 1983.

Justification for classification or non-classification

Based on the available weight of evidence, the test substance, 'potassium lauroyl wheat amino acids', does not warrant classification for acute oral toxicity, according to the EU CLP criteria (Regulation 1272/2008/EC).