Registration Dossier

Administrative data

Description of key information

Overall, although the protein hydrolysates as such are not acutely toxic, their bonding with the quaternary ammonium compound (i.e., Quab 360), which was found to cause acute toxicity and leading to ‘Acute Tox. 4’ classification, is suspected to influence the overall acute toxicity potential of the test substance, as identified in the NRU screening assay. Therefore, as a worst case the LD50 of the test substance, ‘Cocodimonium hydroxypropyl hydrolysed silk’ is expected to lie in the range 300-2000 mg/kg bw (or at 541 mg/kg bw similar to Quab 360).

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Referenceopen allclose all

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 endpoint. Incorporated NR is released from the cells using a solubilisation solution. The absorbance of the NR is quantified using a spectrophotometer.
GLP compliance:
yes (incl. QA statement)
Test type:
other: Neutral Red Uptake (NRU) Cytotoxicity Test Using Human Dermal Fibroblasts in Xeno-Free Culture Conditions
Limit test:
no
Species:
other: Cultured human dermal fibroblasts in animal product-free culture
Strain:
other: Not Applicable
Sex:
not specified
Details on test animals or test system and environmental conditions:
Neonatal human dermal fibroblast cultures - “Xeno-Free” (HDFn-XF) were obtained commercially as cryopreserved primary cells (Lifeline Cell Technology, Carlsbad, USA). They were originally derived from donor tissue with informed consent for the tissue to be used for research purposes, in adherence with the Human Tissue Act (UK) 2004. Xeno-free culture medium and sub-culture reagents (Lifeline Cell Technology, Carlsbad, USA) were free of animal-derived components, providing a fully human cell culture system. Test system was extensively QC tested, by the manufacturer, for a range of parameters including viability upon thawing from cryopreservation, proliferation rate, morphology and sterility (absence of bacteria, fungal growth and mycoplasma). They were also demonstrated to be negative for HIV-1, HIV-2, HBV and HCV.
Route of administration:
other: Refer "Details on oral exposure"
Vehicle:
other: Serum-Free culture medium
Details on oral exposure:
1) Method of administration of test substance
A single application of 8 concentrations of the test substance (n=6) was applied in cell culture medium (dilution factor of 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.
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).
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.

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%.

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.

Endpoint:
acute toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
From March 31, 1981 to April 14, 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category 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:
no guideline followed
Principles of method if other than guideline:
The test substance was administered orally by gavage in graduated doses to several groups of experimental animals, one dose being used per group. Subsequently observations of effects and deaths are made. Animals which die during the test are necropsied, and at the conclusion of the test the surviving animals are sacrificed and necropsied.
GLP compliance:
yes
Test type:
other: Hagan et al 1959 Method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Doses:
5 gm/kg bw
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Mortality:
No moratality was observed.
Body weight:
No significant changes in bodyweight were observed in any animal.
Gross pathology:
No gross changes were observed in all animals.

Results

Animal No and Sex

Bodyweight (gm)

Hours

Days

Bodyweight (gm)

1

3

6

24

2

3

4

5

6

7

8

9

10

11

12

13

14

 

1M

244

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

372

2M

240

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

364

3M

244

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

378

4M

264

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

418

5M

234

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

368

6F

226

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

276

7F

244

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

300

8F

240

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

308

9F

230

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

284

10F

248

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

296

N = Normal

D= Depression

SD = Slight Depression

XD = Severe Depression

Interpretation of results:
other: not classified based on EU CLP criteria
Conclusions:
Based on results of the read across study, the LD50 of the test substance is considered to be >5000 mg/kg bw.
Executive summary:

A study was conducted to determine the acute toxicity potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed keratin' (active content not specified), according to a method described by Hagan et al 1959, in compliance with GLP. Ten albino rats (5 animals each sex), weighing 200 to 300 g, each received a single oral dose of the test substance at a dose level of 5000 mg/kg bw. Animals were observed for pharmacological activity and drug toxicity at 1, 3, 6 and 24 h after treatment and daily thereafter for a total of 14 d. Non-survivors and animals surviving the 14 d observation period were subjected to gross necropsy, with all findings noted. No significant changes in bodyweight or gross were observed in any animal. Under the study conditions, the LD50 of the read across substance was determined to be >5000 mg/kg bw (CPT 1981). Based on results of the read across study, similar oral LD50 can be expected for the test substance, 'Cocodimonium hydroxypropyl hydrolysed silk'.

Endpoint:
acute toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
From January 24, 1983 to February 11, 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category 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:
no guideline followed
Principles of method if other than guideline:
The test substance was administered orally by gavage in graduated doses to several groups of experimental animals, one dose being used per group. Subsequently observations of effects and deaths are made. Animals which die during the test are necropsied, and at the conclusion of the test the surviving animals are sacrificed and necropsied.
GLP compliance:
yes
Test type:
other: Hagan et al 1959 Method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Doses:
5 gm/kg bw
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 5 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: Equivalent to >1000 mg/kg bw a.i.
Mortality:
Only one female animal died on the Day 9 of the observation period.
Body weight:
No significant changes in bodyweight were observed in any animal.
Gross pathology:
No gross changes were observed in nine animals, whereas fibrous tissue encasing heart and lungs was noted in one animal.

Results

Animal No and Sex

Bodyweight (gm)

Hours

Days

Bodyweight (gm)

1

3

6

24

2

3

4

5

6

7

8

9

10

11

12

13

14

 

1M

208

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

300

2M

204

N

N

N

N

N

N

N

N6

N

N6

N

N

N

N

N

N

N

320

3M

222

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

340

4M

222

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

356

5M

206

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

342

6F

210

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

272

7F

238

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

300

8F

220

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

310

9F

236

N

N

N

N

N

N

N

N

N6

SD6,9

+

-

-

-

-

-

-

236

10F

218

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

290

Comments: Animal 1-8, 10: No gross changes observed. 9: Fibrous tissue encasing heart and lungs

N = Normal

D= Depression

SD = Slight Depression

XD = Severe Depression

+ = animal death

1: Hair moist and matted       

2: Hair matted and unkempt       

3: Probable middle ear infection       

4: Diarrhea       

5: Mucoid diarrhea       

6: Appears dehydrated       

7: Convulsions       

8: Muscle tremors       

9: Dried blood around nares

Interpretation of results:
other: EU CLP classification criteria not met
Remarks:
no mortality or systemic toxicity was observed
Conclusions:
Based on results of the read across study, the LD50 of the test substance is considered to be >5000 mg/kg bw (Equivalent to >1000 mg/kg bw a.i.).
Executive summary:

A study was conducted to determine the acute toxicity potential of the read across substance, 'Hydrolysed keratin' (active: 20%), according to a method described by Hagan et al 1959, in compliance with GLP. Ten albino rats (5 animals each sex), weighing 204 to 256 g, each received a single oral dose of the test substance at a dose level of 5000 mg/kg bw. Animals were observed for pharmacological activity and drug toxicity at 1, 3, 6 and 24 h after treatment and daily thereafter for a total of 14 d. Non-survivors and animals surviving the 14 d observation period were subjected to gross necropsy, with all findings noted. No significant changes in bodyweight were observed in any animal. Only one female animal died on the Day 9 of the observation period. No gross changes were observed in nine animals, whereas fibrous tissue encasing heart and lungs was noted in one animal. Under the study conditions, the LD50 of the read across substance was determined to be >5000 mg/kg bw ( i.e., equivalent to >1000 mg/kg bw a.i.) (CPT 1983). Based on results of the read across study, similar absence of mortality or systemic toxicity following acute exposure is expected for the test substance, 'Cocodimonium hydroxypropyl hydrolysed silk'.

Endpoint:
acute toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
From July 07, 1987 to July 21, 1987
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 Category 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:
not specified
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals and Husbandry
Young adult Sprague Dawley derived rats of Crl:CD (SD) BR strain were supplied by Charles River (UK) Limited, Margate, Kent and were delivered by road transport on 19th June 1987. After arrival at the testing facilities animals were permitted an acclimatization period of eighteen days. Animals were housed in single sex groups of five in grid bottomed polypropylene cages . A commercially available pelleted rodent diet (SQC R and M No.1 expanded produced by special diet services, Witham, Essex) and mains drinking water via polypropylene bottles were provided ad libitum. The animal room was illuminated by fluorescent light to give a 24 h cycle of 12 h light/ 12 h dark. The room was air conditioned with the air temperature maintained within the range 18-24ºC and relative humidity within the range 43-76% during the study.



Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
oral gavage
Doses:
10 g/kg bw (neat single dose)
No. of animals per sex per dose:
5 male and 5 female
Control animals:
no
Details on study design:
Dosing
A group of five male and five female animals were administered a single dose level of the test substance. Healthy animals were fasted overnight prior to dosing. Animals were selected for treatment and weighed, the weight being used to calculate the amount of test substance to be administered. This was dosed neat at a dose level of 10g/kg. Animals were dosed by peroral injection using a rubber catheter attached to a syringe of suitable capacity. After dosing animals were returned to their cages end permitted access to rood and water.

Observations
All animals were examined frequently after dosing and then daily for 14 consecutive days. Any signs of toxicity or other effects were noted along with the time of onset and duration. Animals were weighed at weekly intervals.

Necropsy
At the end of the fourteen day post dose observation period surviving animals were weighed and then sacrificed by carbon dioxide asphyxiation. Animals killed thus and those dying during the study were then subjected to gross examination including the opening of the cranial, thoracic and visceral cavities.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
< 10 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: Equivalent to <2500 mg/kg bw a.i.
Mortality:
One male animal was found dead on Day 2, two male and four female animals were found dead on Day 3 and a further male animal was found dead on Day 4. The surviving male animal had completely recovered by Day 4 and the female animal had recovered by Day 5. The test substance had significant toxic effects when administered as a single oral dose at a level of 10 g/kg bw. Observed mortality was eighty percent.
Clinical signs:
Piloerection was observed in all animals within one hour of dosing and was accompanied by red fur staining on Day 2 in male animals and Day 3 in the surviving female animal. Perinasal staining was observed in one female animals by Day 2.
Gross pathology:
No abnormalities were noted at necropsy in the surviving animals. Distension of the gastro-intestinal tract, perinasal, peribuccal and perianal staining were noted in the decedents on necropsy examination. Dark coloration pf lungs, heart and liver was apparent in one animal.

Results

Main study - Individual findings:

Animal No. 1M

Piloerection observed 1 h after dosing accompanied by red fur staining by Day 2. The animal was found dead on Day 2. At necropsy the gastro-intestinal tract was distended with gas and brown fluid.

Animal No. 2M

Piloerection observed 1 h after dosing. This persisted until Day 2 when the animal was found dead. At necropsy perinasal staining and a clear fluid in the stomach were noted.

Animal No. 3M

Piloerection observed 1 h after dosing accompanied by red fur staining by Day 2. The animal was found dead on Day 2. The animal was found dead on Day 3. At necropsy peribuccal staining was noted and the gastro-intestinal tract was distended with gas and brown fluid.

Animal No. 4M

Piloerection observed 1 h after dosing accompanied by red fur staining by Day 2. The animal was found dead on Day 2 and hypoactivity on Day 3. The animal was found dead on Day 4. At necropsy peribuccal staining was noted, the gastro-intestinal tract was distended with gas and brown liquid. Dark coloration of the lungs, heart and liver was noted.

Animal No. 5M

Piloerection observed 1 h after dosing accompanied by red fur staining by Day 2 and 3. Complete recovery occurred by Day 4. No other effects of treatment observed and no abnormalities noted at necropsy.

Animal No. 1F

Piloerection observed 1 h after dosing. Found dead on Day 3. At necropsy peribuccal staining was noted and the gastro-intestinal tract was distended with gas and brown fluid.

Animal No. 2F

Piloerection observed 1 h after dosing. The animal was found dead on Day 3. At necropsy the gastro-intestinal tract was distended with gas and brown fluid.

Animal No. 3F

Piloerection observed 1 h after dosing accompanied by red fur staining by Day 2. The animal was found dead on Day 3. At necropsy the gastro-intestinal tract was distended with gas and brown fluid.

Animal No. 4F

Piloerection observed 1 h after dosing. The animal was found dead on Day 3. At necropsy the gastro-intestinal tract was distended with gas and brown fluid.

Animal No. 5F

Piloerection observed 1 h after dosing accompanied by red fur staining by Day 3. This staining remained until Day 4. Complete recovery occurred by Day 5. No other effects of treatment observed and no abnormalities noted at necropsy.

Bodyweights - Individual values - Main study

Dose level (g/kg)

Animal number

Sex

Bodyweight on Day (g)

 

Change in bodyweight (Day1-15)

1

8

15

 

 

 

 

 

 

 

 

 

 

10 g/kg

 

 

 

 

 

 

 

 

 

 

 

 

D1/1

 

 

 

 

 

 

M

 

 

165

-

-

-

D1/2

162

-

-

-

D1/3

167

-

-

-

D1/4

168

-

-

-

D1/5

156

211

246

90

Mean

164

N/A

N/A

N/A

SD

4.8

N/A

N/A

N/A

D2/1

 

 

 

 

 

F

 

 

 

 

 

132

-

-

-

D2/2

129

-

-

-

D2/3

129

-

-

-

D2/4

132

-

-

-

D2/5

134

174

197

63

Mean

131

N/A

N/A

N/A

SD

2.2

N/A

N/A

N/A

Interpretation of results:
other: not classified based on EU CLP criteria
Conclusions:
Based on results of the read across study, the LD50 of the test substance is considered to be <10 g/kg bw (Equivalent to <2500 mg/kg bw a.i.).
Executive summary:

A study was conducted to determine the acute toxicity potential of the read across substance, 'Hydrolysed keratin from wool' (active: 25%), according to OECD Guideline 401 (limit test), in compliance of GLP. Following overnight fasting, groups of five male and five female rats were administered the test substance at single dose of 10 g/kg bw, by oral gavage route. All animals were observed for a 14 d period for any signs of toxicity or other effects of treatment. Piloerection was observed in all animals within one hour of dosing. One male animal was found dead on Day 2, two male and four female animals were found dead on Day 3 and a further male animal was found dead on Day 4. Abnormalities noted at necropsy were distension of the gastro-intestinal tract, perinasal, peribuccal and perianal staining. Dark coloration pf lungs, heart and liver was apparent in one animal. The test substance had significant toxic effects when administered as a single oral dose at a level of 10 g/kg bw. Observed mortality was 80%. Under the study conditions, the LD50 of the read across substance could not be established and was considered to be <10 g/kg bw (i.e., equivalent to <2500 mg/kg bw a.i.) (TLL, 1987). Based on results of the read across study, similar percentage of mortality at the tested dose of 10 g/kg bw is expected for the test substance, 'Cocodimonium hydroxypropyl hydrolysed silk'.

Endpoint:
acute toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
From December 17, 1987 to December 31, 1987
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 Category 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:
equivalent or similar to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
not specified
GLP compliance:
no
Test type:
other: Hagan et al 1959 Method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals and Husbandry
Young adult Sprague Dawley derived rats of Crl:CD BR strain were supplied by Charles River (UK) limited. Animals were allowed a acclimatisation period of six days. Animals were housed in single sex groups of five in grid bottomed polypropylene cages. A commercially available pelleted rodent diet and mains drinking water via polypropylene bottles were provided ad libitum. The animal room was illuminated by fluorescent light to give a 24 h cycle of 12 h light/12 h dark and the room was air conditioned with the air temperature maintained within the range 19-24ºC and relative humidity within the range 28-67% during the acclimatisation and study periods.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The test substance was bulked in distilled water to give a dose volume of 10 mL/kg bw at dose level of 2000 mg/kg bw.
Doses:
2 gm/kg bw
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: Equivalent to >500 mg/kg bw a.i.
Clinical signs:
Piloerection was apparent in one female rat approximately one hour after dosing.
Body weight:
No significant changes in bodyweight were observed in any animal.
Gross pathology:
Necropsy revealed raddening of the glandular mucosa of the stomach in two female animals. The adrenals of one of these animals also appeared red and the kidneys were mottled and dark in color.
Other findings:
No other signs of toxicity were observed.

Results

Clinical observation

Animal number and Sex

Clinical observations

1M

No signs of toxicity or abnormalities were observed

2M

No signs of toxicity or abnormalities were observed

3M

No signs of toxicity or abnormalities were observed

4M

No signs of toxicity or abnormalities were observed

5M

No signs of toxicity or abnormalities were observed

6F

Piloerection observed one hour after dosing

Stomach glandular mucosa red

7F

No signs of toxicity or abnormalities were observed

8F

No signs of toxicity or abnormalities were observed

9F

No signs of toxicity were observed. Kidneys were dark and mottled. Adrenals were red. Stomach glandular mucosa red

10F

No signs of toxicity or abnormalities were observed

Effect on body weight

Dose level

mg/kg

Animal number and Sex

Bodyweight (g)

Change in bodyweight (g)

 

 

 

 

 

 

 

 

 

 

 

 

2000 mg/kg bw

Day

Day

1

8

15

1-15

1M

114

170

226

112

2M

107

190

235

128

3M

120

183

228

108

4M

103

163

214

111

5M

109

166

208

99

Mean

111

174

222

112

SD

6.6

11.6

11

10.5

6F

122

163

179

57

7F

126

178

196

70

8F

117

165

189

72

9F

125

171

201

76

10F

113

157

184

71

Mean

121

167

190

69

SD

5.5

8

8.9

7.2

Interpretation of results:
other: EU CLP classification criteria not met
Remarks:
(no mortality or systemic toxicity observed)
Conclusions:
Based on results of the read across study, similar absence of toxicity following acute exposure is expected for the the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool'.
Executive summary:

A study was conducted to determine the acute toxicity potential of the read across substance, 'Hydrolysed keratin from wool' (active: 25%), according to OECD Guideline 401 (limit test), in compliance with GLP. Following overnight fasting, groups of five male and five female rats were administered the test substance at 2000 mg/kg bw, by oral gavage route. All animals were observed for a 14 d period for any signs of toxicity or other effects of treatment. One female animal did exhibit piloerection immediately after dosing. No other signs of toxicity were observed and minor abnormalities were noted in two animals at necropsy. Under the study conditions, the oral LD50 of the read across substance was determined to be >2000 mg/kg bw (i.e., equivalent to >500 mg/kg bw a.i.) (TLL, 1988). Based on results of the read across study, similar absence of toxicity following acute exposure is expected for the the test substance, 'Cocodimonium hydroxypropyl hydrolysed silk'.

Endpoint:
acute toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
From January 14, 1987 to February 07, 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category 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:
not specified
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals and Husbandry
Young adult Sprague Dawley derived rats were supplied by A. Smith, Warlingham, Surrey. After arrival at the testing facilities animals were permitted an acclimatization period of at least five days. Animals were housed in single sex groups of five in grid bottomed polypropylene cages. A commercially available pelleted rodent diet (modified 41B supplied by Pillsbury's Limited of Birmingham) and mains drinking water via polypropylene bottles were provided ad libitum. The animal room was illuminated by fluorescent light to give a 24 h cycle of 12 h light/ 12 h dark. The room was air conditioned with the air temperature maintained within the range 16-23ºC and relative humidity within the range 38-91% during the study.


Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
oral gavage
Doses:
10 g/kg bw (neat single dose)
No. of animals per sex per dose:
5 male and 5 female
Control animals:
no
Details on study design:
Dosing
A group of five male and five female animals were administered a single dose level of the test substance. Healthy animals were fasted overnight prior to dosing. Animals were selected for treatment and weighed, the weight being used to calculate the amount of test substance to be administered. This was dosed neat at a dose level of 10g/kg. Animals were dosed by peroral injection using a rubber catheter attached to a syringe of suitable capacity. After dosing animals were returned to their cages end permitted access to rood and water.

Observations
All animals were examined frequently after dosing and then daily for 14 consecutive days. Any signs of toxicity or other effects were noted along with the time of onset and duration. Animals were weighed at weekly intervals.

Necropsy
At the end of the fourteen day post dose observation period all animals were weighed and then sacrificed by carbon dioxide asphyxiation. Animals were then subjected to gross examination including the opening of the cranial, thoracic and visceral cavities.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 10 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality
Clinical signs:
No treatment related effects observed
Body weight:
No treatment related effects observed
Gross pathology:
No treatment related effects observed
Other findings:
No treatment related effects observed

Results

Main study - Individual findings:

Animal No. D1/1M

No effects of treatment observed during the study and no abnormalities noted at necropsy

Animal No. D1/2M

No effects of treatment observed during the study and no abnormalities noted at necropsy

Animal No. D1/3M

No effects of treatment observed during the study and no abnormalities noted at necropsy

Animal No. D1/4M

No effects of treatment observed during the study and no abnormalities noted at necropsy

Animal No. D1/5M

No effects of treatment observed during the study and no abnormalities noted at necropsy

Animal No. D2/1F

No effects of treatment observed during the study and no abnormalities noted at necropsy

Animal No. D2/2F

No effects of treatment observed during the study and no abnormalities noted at necropsy

Animal No. D2/3F

No effects of treatment observed during the study and no abnormalities noted at necropsy

Animal No. D2/4F

No effects of treatment observed during the study and no abnormalities noted at necropsy

Animal No. D2/5F

No effects of treatment observed during the study and no abnormalities noted at necropsy

Bodyweights - Individual values - Main study

Dose level (g/kg)

Animal number

Sex

Bodyweight on Day (g)

Change in bodyweight (Day1-15)

1

8

15

 

 

 

 

 

 

 

 

 

10 g/kg

D1/1

 

 

 

 

 

M

327

409

456

129

D1/2

324

407

414

90

D1/3

312

384

423

111

D1/4

351

435

485

134

D1/5

340

431

451

139

Mean

331

413

479

121

SD

15.1

20.6

32.1

20.1

D2/1

 

 

 

 

F

259

271

289

30

D2/2

266

291

330

64

D2/3

264

290

368

104

D2/4

260

267

274

14

D2/5

260

266

268

8

Mean

262

277

306

44

SD

3

12.5

42.4

40

Interpretation of results:
other: EU CLP classification criteria not met
Remarks:
no mortality or systemic toxicity was observed
Conclusions:
Based on results of the read across study, the LD50 of the read across substance is considered to be >10 g/kg bw.
Executive summary:

A study was conducted to determine the acute toxicity potential of the read across substance, 'Hydrolysed keratin' (active content not specified), according to OECD Guideline 401 (limit test), in compliance with GLP. Following overnight fasting, groups of five male and five female rats were administered single dose of 10 g/kg bw test substance, by oral gavage route. All animals were observed for a 14 d period for any signs of toxicity or other effects of treatment. No treatment related adverse effects or signs of toxicity and no abnormalities at necropsy were noted in all animals. No significant body weight change related to treatment was observed. Under the study conditions, the oral LD50 of the read across substance was determined to be >10 g/kg bw (TLL, 1986). Based on results of the read across study, similar absence of mortality or systemic toxicity following acute exposure is expected for the test substance, 'Cocodimonium hydroxypropyl hydrolysed silk'.

Endpoint:
acute toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
Refer to section 13 of IUCLID for details on the Category 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:
not specified
GLP compliance:
not specified
Species:
rat
Strain:
Wistar
Remarks:
Bor: WISW
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
other: aqua dest.
Details on oral exposure:
The test was performed with undiluted test substance.
Doses:
Male: 389, 573, 693, 838 mg/kg
Female: 1015, 1230, 1806 mg/kg
No. of animals per sex per dose:
5
Control animals:
not specified
Details on study design:
The test was performed with undiluted test substance. Four groups of 5 male rats were dosed with 693, 838, 1015, 1230 mg/kg of the test substance. Three groups of 5 females were dosed with 389, 573, 838 mg/kg of the test substance.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
541 other: mg/kg
Based on:
act. ingr.
95% CL:
> 354 - < 827
Remarks on result:
other: corrected for 35% purity
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
912 other: mg/kg
Based on:
act. ingr.
95% CL:
> 742 - < 1 121
Remarks on result:
other: corrected for 35% purity

Signs of toxicity related to dose level used:

Piloerection, restrained gait, stilted gait, diarrhea, sunken sides, body weight decrease, individual chromodacryorhoea, and tremor, premortal general loss of reflexes. First signs of intoxication symptoms were seen 2 to 5 hours p. a. in the low dose 1 to 2 days, death between day 1 and 4.

Effects on organs (related to dose level): mucous membrane of the stomach and duodenum dark-red coloured. Stomach tympanic, stomach and abdomen filled with liquid.

Interpretation of results:
other: Category 4 based on CLP criteria
Conclusions:
Based on results of the read across study, the oral LD50 of the test substance is considered at 541.0 mg/kg bw (95% confidence intervals: 354.0 -827.0) for female rats and at 912 mg/kg bw (95% confidence intervals: 742 -1121) for male rats.
Executive summary:

A study was conducted to determine the acute toxicity potential of the read across substance, 'Quab 360' (active: 35%) when administered by oral gavage to Wistar rats (Bor: WISW) according to OECD Guideline 401. The test was performed with undiluted substance. Four groups of 5 male rats were dosed with 693, 838, 1015, 1230 mg a.i./kg bw and three groups of 5 females were dosed with 389, 573, 838 mg/kg bw of the read across substance; doses were corrected for the 35% active content of the read across substance. Following signs of toxicity were noted: piloerection, restrained gait, stilted gait, diarrhea, sunken sides, body weight decrease, individual chromodacryorrhoea, and tremor, premortal general loss of reflexes. First signs of intoxication symptoms were seen 2 to 5 h post administration and in the low dose 1 to 2 d post administration. Mortality was observed between Day 1 and 4. Gross pathological findings showed red-coloured mucous membrane of the stomach and duodenum. Stomach tympanic and stomach and abdomen filled with liquid. Under the study conditions, the oral LD50 of the read across substance was determined at 541 mg/kg bw (95% confidence intervals: 354 -827) for female rats and at 912 mg/kg bw (95% confidence intervals: 742 -1121) for male rats (Degussa, 2007). Based on results of the read across study, similar levels of mortality and oral LD50 value is expected for the test substance, 'Cocodimonium hydroxylpropyl hydrolysed silk'.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
541 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

The acute toxicity potential of the test substance has been assessed based on studies of in vitro study with the test substance as well as substances representative of the overall substance and the 2 main components used to manufacture the test substance, which can be categorised as hydrolysed proteins and quaternary ammonium compound (i.e., Quab 360). The results are presented below:

In vitro study with the test substance

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 (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 (i.e., 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 read across 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.

In vivo study with the read across substance:

A study was conducted to determine the acute toxicity potential of the read across substance, 'Cocodimonium hydroxypropyl hydrolysed keratin' (active content not specified), according to a method described by Hagan et al 1959, in compliance with GLP. Ten albino rats (5 animals each sex), weighing 200 to 300 g, each received a single oral dose of the test substance at a dose level of 5000 mg/kg bw. Animals were observed for pharmacological activity and drug toxicity at 1, 3, 6 and 24 h after treatment and daily thereafter for a total of 14 d. Non-survivors and animals surviving the 14 d observation period were subjected to gross necropsy, with all findings noted. No significant changes in bodyweight or gross were observed in any animal. Under the study conditions, the LD50 of the read across substance was determined to be >5000 mg/kg bw (CPT 1981).

In vivo studies with 2 main components used to manufacture the test substance:

Hydrolysed proteins:

Study 1:A study was conducted to determine the acute toxicity potential of the read across substance, 'Hydrolysed keratin (active: 20%), according to a method described by Hagan et al 1959, in compliance with GLP. Ten albino rats (5 animals each sex), weighing 204 to 256 g, each received a single oral dose of the test substance at a dose level of 5000 mg/kg bw. Animals were observed for pharmacological activity and drug toxicity at 1, 3, 6 and 24 h after treatment and daily thereafter for a total of 14 d. Non-survivors and animals surviving the 14 d observation period were subjected to gross necropsy, with all findings noted. No significant changes in bodyweight were observed in any animal. Only one female animal died on the Day 9 of the observation period. No gross changes were observed in nine animals, whereas fibrous tissue encasing heart and lungs was noted in one animal. Under the study conditions, the LD50 of the read across substance was determined to be >5000 mg/kg bw ( i.e., equivalent to >1000 mg/kg bw a.i.) (CPT 1983).

Study 2:A study was conducted to determine the acute toxicity potential of the read across substance, 'Hydrolysed keratin (active content not specified)', according to OECD Guideline 401 (limit test), in compliance with GLP. Following overnight fasting, groups of five male and five female rats were administered single dose of 10 g/kg bw test substance, by oral gavage route. All animals were observed for a 14 d period for any signs of toxicity or other effects of treatment. No treatment related adverse effects or signs of toxicity and no abnormalities at necropsy were noted in all animals. No significant body weight change related to treatment was observed. Under the study conditions, the oral LD50 of the read across substance was determined to be >10 g/kg bw (TLL, 1986).

Study 3:A study was conducted to determine the acute toxicity potential of the read across substance, 'Hydrolysed keratin from wool (active: 25%)', according to OECD Guideline 401 (limit test), in compliance with GLP. Following overnight fasting, groups of five male and five female rats were administered the test substance at 2000 mg/kg bw, by oral gavage route. All animals were observed for a 14 d period for any signs of toxicity or other effects of treatment. One female animal did exhibit piloerection immediately after dosing. No other signs of toxicity were observed and minor abnormalities were noted in two animals at necropsy. Under the study conditions, the oral LD50 of the read across substance was determined to be >2000 mg/kg bw (i.e., equivalent to >500 mg/kg bw a.i.) (TLL,1988).

Further, the hydrolysed proteins differences across the read across substances is not expected to have an impact, as the proteins in general are not toxic and form and play an important part in the living organisms (such as enzymes, antibodies, hormones, transport or structural proteins, essential elements of the motile and contractile systems) (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. Moreover, 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.

Quaternary ammonium compounds (QAC) – Quab 360

A study was conducted to determine the acute toxicity potential of the read across substance, Quab 360 (active: 35%) when administered by oral gavage to Wistar rats (Bor: WISW) according to OECD Guideline 401. The test was performed with undiluted substance. Four groups of 5 male rats were dosed with 693, 838, 1015, 1230 mg a.i./kg bw and three groups of 5 females were dosed with 389, 573, 838 mg/kg bw of the read across substance; doses were corrected for the 35% active content of the read across substance. Following signs of toxicity were noted: piloerection, restrained gait, stilted gait, diarrhea, sunken sides, body weight decrease, individual chromodacryorrhoea, and tremor, premortal general loss of reflexes. First signs of intoxication symptoms were seen 2 to 5 h post administration and in the low dose 1 to 2 d post administration. Mortality was observed between Day 1 and 4. Gross pathological findings showed red-coloured mucous membrane of the stomach and duodenum. Stomach tympanic and stomach and abdomen filled with liquid. Under the study conditions, the oral LD50 of the read across substance was determined at 541 mg/kg bw (95% confidence intervals: 354 -827) for female rats and at 912 mg/kg bw (95% confidence intervals: 742 -1121) for male rats (Degussa, 2007).

Overall, although the protein hydrolysates as such are not acutely toxic, their bonding with the quaternary ammonium compound, which was found to cause acute toxicity and leading to ‘Acute Tox. 4’ classification is suspected to influence the overall acute toxicity potential of the test substance, as identified in the NRU assay with the read across substance. Therefore, as a worst case the LD50 of the test substance, ‘Cocodimonium hydroxypropyl hydrolysed silk’ is expected to lie in the range 300-2000 mg/kg bw.

References:

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

Justification for classification or non-classification

Based on the weight of evidence information, the test substance, 'Cocodimonium hydroxypropyl hydrolysed silk' is concluded to warrant 'Acute Tox.4, H302: Harmful if swallowed' classification according to EU CLP criteria (Regulation EC 1272/2008).