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EC number: 934-512-9 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
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
- 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
- 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.
- 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'.
Referenceopen allclose all
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.
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).
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