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EC number: 947-921-2 | 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
Acute Toxicity: oral
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 129: Guidance document on using cytotoxicity tests to estimate starting doses for acute oral systemic toxicity tests
- Deviations:
- not specified
- Principles of method if other than guideline:
- The Neutral Red Uptake (NRU) assay is used to determine cell viability as an indicator of acute toxicity. Neutral Red (a weak cationic dye), penetrates cellular membranes, entering cells via non-ionic diffusion and accumulates intracellularly in lysosomes. Viable cells take up and retain the Neutral Red (NR) dye, while damaged or dead cells do not, therefore, the Neutral Red Uptake (NRU) assay can be employed as a direct measure of cell viability, using membrane integrity as the measured endpoint. Incorporated NR is released from the cells using a solubilisation solution. The absorbance of the NR is quantified using a spectrophotometer.
- GLP compliance:
- yes (incl. QA statement)
- Test type:
- other: Neutral Red Uptake (NRU) Cytotoxicity Test Using Human Dermal Fibroblasts in Xeno-Free Culture Conditions
- Limit test:
- no
Test material
- Reference substance name:
- Protein hydrolyzates, silk, reaction products with 3-chloro-2-hydroxypropyl-cocoalkyl-dimethylammonium chloride
- Molecular formula:
- Molecular formula of major active constituents: C20H43CL1N2O3 (representative molecular formula for C12 alkyl chain Quaternised alanine) C19H41CL1N2O3 (representative molecular formula for C12 alkyl chain Quaternised glycine) C20H43Cl1N2O4 (representative molecular formula of C12 alkyl chain quaternised serine)
- IUPAC Name:
- Protein hydrolyzates, silk, reaction products with 3-chloro-2-hydroxypropyl-cocoalkyl-dimethylammonium chloride
- Test material form:
- other: Paste
Constituent 1
Test animals
- Species:
- other: Cultured human dermal fibroblasts in animal product-free culture
- Strain:
- other: Not Applicable
- Sex:
- not specified
- Details on test animals or test system and environmental conditions:
- Neonatal human dermal fibroblast cultures - “Xeno-Free” (HDFn-XF) were obtained commercially as cryopreserved primary cells (Lifeline Cell Technology, Carlsbad, USA). They were originally derived from donor tissue with informed consent for the tissue to be used for research purposes, in adherence with the Human Tissue Act (UK) 2004. Xeno-free culture medium and sub-culture reagents (Lifeline Cell Technology, Carlsbad, USA) were free of animal-derived components, providing a fully human cell culture system. Test system was extensively QC tested, by the manufacturer, for a range of parameters including viability upon thawing from cryopreservation, proliferation rate, morphology and sterility (absence of bacteria, fungal growth and mycoplasma). They were also demonstrated to be negative for HIV-1, HIV-2, HBV and HCV.
Administration / exposure
- Route of administration:
- other: Refer "Details on oral exposure"
- Vehicle:
- other: Serum-Free culture medium
- Details on oral exposure:
- 1) Method of administration of test substance
A single application of 8 concentrations of the test substance (n=6) was applied in cell culture medium (dilution factor of 5 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): 200000, 40000, 8000, 1600, 320, 64, 12.8, 2.56
Main experiments (µg/mL): 300, 200, 133.3, 88.9, 59.3, 39.5, 26.3, 17.6
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: 30 Sep 17 for ME1, ME2, ME3, Sep 18 for RFE)
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): 200000, 40000, 8000, 1600, 320, 64, 12.8, 2.56 µg/mL (dilution factor 5)
Main experiment (ME): 300, 200, 133.3, 88.9, 59.3, 39.5, 26.3, 17.6 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 5 was used) to determine a top concentration for three main experiments (ME) allowing the determination of a more accurate IC50. - No. of animals per sex per dose:
- 6 replicates for test substance and positive control
12 replicates for negative control - Control animals:
- other: culture medium
- Details on study design:
- Overview
Preliminary testing: Determination of the top concentration by solubility testing
Range finding experiment (RFE): To determine a top concentration for the main experiment.
Main experiment (ME) x 3:
Day 1: Seeding cells (1 x 96-well plates for RFE; 3 x 96-well plate for ME).
Day 2: 24 h after seeding, apply test and reference substances for 24 ± 1h
Day 3: Evaluate the Neutral Red Uptake - Statistics:
- Data Analysis for this study were performed following XCellR8 SOP L0064: “Neutral Red Uptake (NRU) Cytotoxicity Test Using Human Dermal Fibroblasts in Xeno-Free Culture Conditions”, using XCellR8 Form F0058: Acute Toxicity Analysis Spreadsheet v01, for processing. This is a Microsoft Excel workbook (created during the project funded by Innovate UK (project number 131726) and validated in-house in August 2017, containing formulae to process the raw data as per SOP L0064. The final data output is a percentage viability value for cells exposed to the test substance relative to the negative control and the IC50 value.
Results and discussion
- Preliminary study:
- As per OECD guidance document 129, an initial range finding experiment (RFE) was performed with a range of concentrations based on the outcome of the solubility test (dilution factor of 5 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 200 mg/mL (200,000 µg/mL).
Effect levelsopen allclose all
- Key result
- Dose descriptor:
- other: IC50
- Remarks:
- the time taken to reduce cell viability to 50% of the negative control
- Effect level:
- ca. 25.3 - ca. 40.5 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
- Key result
- Dose descriptor:
- other: IC50
- Remarks:
- the time taken to reduce cell viability to 50% of the negative control
- Effect level:
- ca. 12.903 - ca. 20.655 other: µg/mL
- Based on:
- act. ingr.
- Remarks on result:
- other: Equivalent predicted LD50: 300-2000 mg/kg bw
- Remarks:
- Potential EU CLP classification: Category 4
- Other findings:
- The IC50 value obtained in all experiments were between 10-1000 µg/mL, therefore, the test substance was classified as GHS Category 4 “Harmful if swallowed”, suggesting a low acute toxicity potential.
Any other information on results incl. tables
Results
Solubility Results
The solubility was first determined following OECD guidance document 129 to determine the top concentration for the RFE:
Tier 1: 200mg/mL in cell culture medium - SOLUBLE
Therefore the top concentration used in the range finding experiment (RFE) described here after was 200 mg/mL (200,000 µg/mL).
Range Finding Experiment
An initial Range Finding Experiment (RFE) was performed where a top concentration of 200,000 µg/mL (200 mg/mL) of the test substance was used, as described above. The dilution factor was 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-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 IC50was 58.3 µg/mL was 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
TA2-RFE |
NC1 |
C1 |
C2 |
C3 |
C4 |
C5 |
C6 |
C7 |
C8 |
NC2 |
Concentration (µg/mL) |
0.0 |
200000 |
40000 |
8000 |
1600 |
320 |
64 |
12.8 |
2.6 |
0.0 |
% of Negative Control |
13.5% |
-1.2% |
5.2% |
9.9% |
-0.1% |
19.1% |
0.2% |
156.8% |
173.3% |
186.5% |
SD |
28.7% |
1.1% |
6.7% |
11.2% |
8.0% |
15.5% |
2.6% |
5.8% |
9.9% |
6.9% |
% CV |
212.42% |
-95.93% |
130.51% |
113.13% |
-7054.08% |
81.09% |
1150.65% |
3.71% |
5.69% |
3.71% |
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. *SD value above 15%, a maximum of 2 outliers were removed for the final calculation. It should be noted that the percentage of viability value of the negative control one (NC1) was below 50%, possibly due to volatility of the test substance. Therefore, the NC1 value was excluded from calculation. Final results are presented in Table 3.
TA2-RFE |
NC1 |
C1 |
C2 |
C3 |
C4 |
C5 |
C6 |
C7 |
C8 |
NC2 |
Concentration (µg/mL) |
0.0 |
200000 |
40000 |
8000 |
1600 |
320 |
64 |
12.8 |
2.6 |
0.0 |
% of Negative Control |
-0.6% |
2.8% |
5.3% |
-0.1% |
10.3% |
0.1% |
84.1% |
92.9% |
100.0% |
|
SD |
0.6% |
3.6% |
6.0% |
4.3% |
8.3% |
1.4% |
3.1% |
5.3% |
3.7% |
|
% CV |
-95.93% |
130.51% |
113.13% |
-7054.08% |
81.09% |
1150.65% |
3.71% |
5.69% |
3.71% |
Table 3: Cell viability measurements 24 h ± 1 h after application of the test substance. NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8. Final results are presented here.
The calculated IC50 was 33.6 µg/mL.
Main Experiments
Three Main Experiments were performed, with a top test substance concentration of 300 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 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: * For SD value above 15%, a maximum of 2 outliers were removed for the final calculation. Final results are presented in Table 5.
PC-ME1 |
NC1 |
C1 |
C2 |
C3 |
C4 |
C5 |
C6 |
C7 |
C8 |
NC2 |
Concentration (µg/mL) |
0.0 |
100.0 |
83.3 |
69.4 |
57.9 |
48.2 |
40.2 |
33.5 |
27.9 |
0.0 |
% of Negative Control |
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 5: Cell viability measurements 24 h ± 1 h after application of positive control (SDS). NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8. A 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 IC50was 45.4 µg/mL was within the range of the historical data obtained during theproject funded by Innovate UK (project number 131726) in which the assay was set up (range 26.5-70.9 µg/mL).
Test substance-ME1
TA2-ME1 |
NC1 |
C1 |
C2 |
C3 |
C4 |
C5 |
C6 |
C7 |
C8 |
NC2 |
Concentration (µg/mL) |
0.0 |
300.0 |
200.0 |
133.3 |
88.9 |
59.3 |
39.5 |
26.3 |
17.6 |
0.0 |
% of Negative Control |
102.2% |
13.9% |
13.1% |
14.3% |
24.2% |
42.2% |
50.4% |
61.1% |
73.6% |
97.8% |
SD |
5.3% |
3.5% |
11.0% |
7.9% |
5.0% |
8.4% |
11.5% |
10.9% |
9.7% |
12.1% |
% CV |
5.16% |
25.07% |
83.67% |
55.09% |
20.47% |
19.95% |
22.75% |
17.78% |
13.16% |
12.42% |
Table 6: Cell viability measurements 24 h ± 1 h after application of the test substance. NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8.
The calculated IC50was 40.5 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 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. * For SD value above 15%, a maximum of 2 outliers were removed for the final calculation. Final results are presented in Table 8.
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 8: Cell viability measurements 24 h ± 1 h after application of positive control (SDS). NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8. A 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 doesn’t impact on the IC50calculation.
The calculated IC50was 37.8 µg/mL was 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-ME2
TA2-ME2 |
NC1 |
C1 |
C2 |
C3 |
C4 |
C5 |
C6 |
C7 |
C8 |
NC2 |
Concentration (µg/mL) |
0.0 |
300.0 |
200.0 |
133.3 |
88.9 |
59.3 |
39.5 |
26.3 |
17.6 |
0.0 |
% of Negative Control |
96.8% |
7.8% |
3.9% |
6.0% |
17.2% |
29.3% |
36.1% |
48.0% |
65.1% |
103.2% |
SD |
9.2% |
9.1% |
8.9% |
2.4% |
4.4% |
5.4% |
8.9% |
10.4% |
6.8% |
9.6% |
% CV |
9.46% |
117.33% |
230.55% |
39.94% |
25.71% |
18.35% |
24.63% |
21.74% |
10.38% |
9.34% |
Table 9: Cell viability measurements 24 h ± 1 h after application of the test substance. NC1 and 2: negative control (untreated cells), C1 to C8: Concentration 1 to 8.
The calculated IC50was 25.3 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 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.
The calculated IC50was 43.1 µg/mL was within the range of the historical data obtained during the project funded by Innovate UK (project number 131726) in which the assay was set up (range 26.5-70.9 µg/mL).
Test substance-ME3
TA2-ME3 |
NC1 |
C1 |
C2 |
C3 |
C4 |
C5 |
C6 |
C7 |
C8 |
NC2 |
Concentration (µg/mL) |
0.0 |
300.0 |
200.0 |
133.3 |
88.9 |
59.3 |
39.5 |
26.3 |
17.6 |
0.0 |
% of Negative Control |
105.3% |
16.0% |
11.4% |
16.0% |
12.1% |
17.2% |
47.3% |
59.8% |
68.7% |
94.7% |
SD |
7.8% |
10.0% |
7.9% |
5.6% |
4.5% |
8.3% |
14.5% |
18.0% |
13.7% |
4.7% |
% CV |
7.44% |
62.58% |
69.74% |
34.77% |
37.38% |
48.23% |
30.72% |
30.11% |
19.88% |
4.94% |
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. For SD value above 15%, a maximum of 2 outliers were removed for the final calculation. Final results are presented in Table 12.
TA2-ME3 |
NC1 |
C1 |
C2 |
C3 |
C4 |
C5 |
C6 |
C7 |
C8 |
NC2 |
Concentration (µg/mL) |
0.0 |
300.0 |
200.0 |
133.3 |
88.9 |
59.3 |
39.5 |
26.3 |
17.6 |
0.0 |
% of Negative Control |
105.3% |
16.0% |
11.4% |
16.0% |
12.1% |
17.2% |
47.3% |
49.6% |
68.7% |
94.7% |
SD |
7.8% |
10.0% |
7.9% |
5.6% |
4.5% |
8.3% |
14.5% |
10.6% |
13.7% |
4.7% |
% CV |
7.44% |
62.58% |
69.74% |
34.77% |
37.38% |
48.23% |
30.72% |
21.44% |
19.88% |
4.94% |
Table 12: 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 IC50calculation. Final results are presented here.
The calculated IC50was 26.1 µg/mL.
Assay acceptance criteria
Results were checked against the following acceptance criteria:
1) Each run includes a Positive Control (SDS) plate with a defined series of concentrations to determine the IC50. In order for the run to be valid, the IC5 0for SDS must be within the mean ± 1.5 SD of the historical set of runs with this substance (48.7 µg/mL ± (1.5x14.8)) - For the RFE, and the 3 ME, the IC50 values obtained with the PC were 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.
2) SD (Standard Deviation) of the 6 values for each condition should be ≤15% (when viability percentage is above 30%) - In some cases, a maximum of 2 outliers was removed to achieve SD ≤15%.
Applicant's summary and conclusion
- Interpretation of results:
- study cannot be used for classification
- Conclusions:
- Under the study conditions, the test substance predicted LD50 was considered to be 300 to 2000 mg/kg bw (Based on in vitro experimental IC50: 12.90 to 20.66 µg/mL).
- Executive summary:
An in vitro study was conducted to determine the acute toxicity potential of test substance, 'Cocodimonium hydroxypropyl hydrolysed silk (active: 51%)', 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 (300, 200, 133.3, 88.9, 59.3, 39.5, 26.3, 17.6 µg/mL) of test substance in cell culture medium of Human Dermal Fibroblasts neonatal (HDFn), cytotoxicity was evaluated. Using a prediction model, determined previously, the IC50 value was converted to a corresponding GHS classification for oral acute toxicity. The 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 33.6 µg/mL (17.14 µg a.i./mL) in the Range Finding Experiment (RFE); 40.5 µg/mL (20.66 µg a.i./mL) in the main experiment 1 (ME1); 25.3 µg/mL (12.90 µg a.i./mL) in ME2 and 26.1 µg/mL (13.31 µg a.i./mL) in ME3. Based on the study results (IC50: 12.90 to 20.66 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.
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