Registration Dossier

Administrative data

Description of key information

Based on the aboveweight of evidence, the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool', is considered to be non-sensitising to the skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From August 30, 2017 to August 31, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Test and Reference substances

Test substance
Concentration tested: 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, 0.098, 0.049, 0.024* µg/mL
Solvent: 1% Ethanol in cell culture medium
Purity: 62.8%
* Concentrations used during the repeat of the study

Positive control
Substance name: Cinnamic Aldehyde
Concentration tested: 8µM to 128 µM
Solvent: 1% Ethanol in cell culture medium
Purity: ≥99%

Negative control
Substance name: Ethanol
Concentration tested: 1%
Purity: ≥99.5%


Key result
Parameter:
other: EC1.5 value
Run / experiment:
All concentrations tested
Value:
< 1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Imax
Remarks:
Maximum-fold induction observed within the concentration range tested
Run / experiment:
0.391µg/mL concentration
Value:
ca. 0.986
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Based on the study results, all of the formal acceptance criteria of the tests were met apart from acceptance criterion 2. However, as all other acceptance criteria were met and there was a dose-dependent increase of induction with the positive control, therefore, results were considered as valid by study author.

Results

Solubility

The test concentrations of test substance used in the KeratinoSensTM test were selected on the basis of solubility test carried out prior to the study. Solubility of test substance in cell culture medium (confirmed up to 200 mg/mL); subsequent dilution in cell culture medium 1% Ethanol giving a top concentration of 100,000µg/mL, a first run with 3 repetitions was performed with the following concentrations: 100,000-50,000-25,000-12,500-6250-3125-1562.5-781.3-390.6-195.3-97.7-48.8 µg/mL. However, all concentrations were cytotoxic (percentage of viability were below 50%). As no interpretation was possible, the study was repeated using a lower top concentration (50µg/mL).The results of the first run are retained in the study data.

 

Table 1: Determination of the skin sensitisation potential of test substance

 

Test substance concentration (µg/mL)

0.024

0.049

0.098

0.195

0.391

0.781

1.563

3.125

6.25

12.5

25

50

Mean of fold induction

0.868

0.875

0.910

0.935

0.986

0.907

0.840

0.786

0.717

0.615

0.622

-0.002

SD

0.109

0.081

0.082

0.181

0.131

0.109

0.106

0.089

0.075

0.115

0.085

0.006

Viability %

102.38

122.59

112.86

116.22

109.17

108.81

103.73

95.16

102.47

99.40

49.71

1.99

Imax

0.986 at 0.391µg/mL

EC1.5

No EC1.5 as no concentration did induce the luciferase activity above the 1.5 threshold

IC50

25 µg/mL

IC30

20 µg/mL

 

Table 2: Determination criteria for the skin sensitisation potential of the test substance

Determination criteria for the skin sensitisation potential of the test substance

 

REP1

REP2

REP3

Does at least one concentration of Test substanceinduce luciferase activity >1.5 -fold:

No

No

No

Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:

N/A

N/A

N/A

Does EC1.5 value occur at a concentration <1000µM (or <200µg/mL)

N/A

N/A

N/A

Does the test substance induce the luciferase in a dose-dependent manner

N/A

N/A

N/A

Classification

Non-sensitiser

Non-sensitiser

Non-sensitiser

 

Table 3: Assay Acceptance Criteria (Mean of the 3 repetitions)

Criteria

Result

Pass or Fail

1 - Positive Control (PC) (Cinnamic aldehyde) induction >1.5-fold in at least one concentration 

Yes

Pass

2 - Average induction of PC at 32µM is [1.6-3.0]

No (1.53)

Fail*

3 - EC1.5value is [6-39µM]

31µM

Pass

4 - CV% of blank values < 20%

Yes (12.2)

Pass

*Acceptance Criterion 2 is not met, however, all other acceptance criteria are met and there is dose-dependent increase of induction with the Positive Control. Therefore, the results are considered as valid.

Interpretation of results:
other: Not classified based on EU CLP criteria
Conclusions:
Under the study conditions, the test substance was considered to be non sensitising to skin.
Executive summary:

An in vitro study was conducted to determine skin sensitisation potential of the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool' (active: 62.8%) using ARE-Nrf2 Luciferase test method, according to OECD Guideline 442D, in compliance with GLP. A single application of 12 concentrations of the test substance (i.e., 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, 0.098, 0.049, 0.024 µg/mL) and 5 concentrations of the positive control (ranging from 8µM to 128 µM) were applied in cell culture medium (dilution factor of 2) with a final concentration of Ethanol of 1%. A single application of culture medium with 1% Ethanol was applied as the negative control. After 24 h, cells were incubated with the test or reference substance for 48 ± 2h at 37C, 5% CO2, ≥ 95% relative humidity before endpoints measurements. Three repetitions were performed and each repetition consisted of 3 x 96-well plates for luminescence and 2x 96-well plates for MTT. The sensitisation potential of the test substance was quantified by calculating two parameters known as the EC1.5 (i.e., effective concentration of test substance that caused an induction of luciferase activity of greater than 1.5-fold over untreated controls) and Imax value (i.e., maximum-fold induction observed within the concentration range tested). The validity of each repetition was assessed following acceptance criteria. In each repetition of the study, no concentration of the test substance caused luciferase induction ≥1.5. Therefore, the test substance was classified as non-sensitising. The maximum induction was observed at a test concentration of 0.391 µg/mL, which showed an Imax value of 0.986. For reference, during test validation, sensitising proficiency chemicals produced Imax values of up to 36-fold over untreated controls. All of the formal acceptance criteria of the tests were met apart from acceptance criterion 2. However, as all other acceptance criteria were met and there was a dose-dependent increase of induction with the positive control, results were considered as valid by the study author. Under the study conditions, the test substance was considered to be non sensitising to skin (XcellR8, 2017).

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 30, 2018 to June 13, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Version / remarks:
OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 29 Jul 2016.
Deviations:
no
Qualifier:
according to
Guideline:
other: EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT)
Version / remarks:
EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT); Skin Sensitisation and Allergic Contact Dermatitis (Issued: 01 Jul 2015)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
OECD TG 442E cites the h-CLAT model using THP-1 cells as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.
Details on study design:
Description of the test method
Skin sensitisers have been reported to induce the expression of cell membrane markers associated with Dendritic Cell (DC) activation. The h-CLAT method is an in vitro assay that quantifies these changes in cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells (a cell line that mimics DCs), following 24-h exposure to the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface maker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

Characterisation of the test method
The h-CLAT has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Method workflow summary
Solubility was first determined for the test substance using either culture medium (RPMI 1640) or DMSO. Note that for this method, test substances with a Log Kow (octanol/water partition coefficient) of up to 3.5 have been tested successfully. However, test substances with a Log Kow of greater than 3.5 can still be tested at lower soluble concentrations. In such a case, a negative result (non-sensitiser) should be considered inconclusive, whereas a positive result (sensitiser) could still be considered valid. THP-1 cells were pre-cultured for 72 ±2 h. Following this, the cells were dosed with the test substance over an 8 dose range and incubated for 24 ±0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The dose of test substance that yields 75% cell viability (CV75) was calculated and taken forward for the next stage of testing. This dose finding assay was carried out over two independent runs. THP-1 cells were pre-cultured for either 48 ±2 or 72 ±2 h. Once the CV75 was determined, a narrower dilution series based around the CV75 value was produced for the test substance. This dilution range was used to dose the cells again for 24 ±0.5 h. The cells were then washed and stained with propidium iodide and also with antibodies that detect CD54 and CD86 expression as well as a negative control antibody. This allowed for discrimination of live/dead cells and also changes in CD54 and CD86 marker expression by flow cytometry.

Test system material source
The human monocytic leukaemia cell line, THP-1, (Cat# TIB-202) were obtained from American Type Culture Collection (ATCC) via their UK subsidiary LGC standards; LGC Standards, Queens Road, Teddington, Middlesex TW11 0LY, UK. E-mail: atcc@lgcstandards.com. Tel: +44 (0)20 8943 8489. Two initial vials of cells were received in April 2016 and data to verify the referenced sources was archived..

OECD test Guideline relating to the test method
OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 09 Oct 2017.

XCellR8 SOP
The study was performed using the h-CLAT method as detailed in OECD TG 442E and also in EURL ECVAM DB-ALM Protocol No. 158 (Issued 02 Mar 2017). Methodology is detailed in an XCellR8 SOP (L0094) that covers the h-CLAT. Study data is collected using XCellR8 internal protocols (IPs).
Key result
Parameter:
other: CV75 (µg/mL)
Remarks:
A concentration showing 75% of THP-1 cell survival (25% cytotoxicity)
Run / experiment:
Average of two run
Value:
ca. 167.6
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: CD54 expression measured as RFI (up to 201.12 µg/mL, top dose concentration)
Run / experiment:
Average of two representatives
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: CD86 expression measured as RFI (up to 201.12 µg/mL, top dose concentration)
Run / experiment:
Average of two representatives
Value:
> 150
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression.

Results

 

a) Solvent Selection and CV75 Determination

Prior to the CV75 determination, the test substance was assessed for solubility and was found to be soluble in complete RPMI culture medium at 250 mg/mL. The CV75 value derived from two independent experiments was as follows:

CV75 (Rep1)

CV75 (Rep2)

Average CV75 (µg/mL)

123.7

211.6

167.6

 

b) Acceptance Criteria

Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Note that the cell viability in the medium control in Run 2 for the CD54/86 measurements was a borderline value (86.61%) and was deemed acceptable even though it fell below the acceptance range for the control.

CV75 Determination

Criterion

Run 1

Run 2

Outcome

Cell viability must be ≥ 75% at the lowest dose.

95.24

96.98

PASS

The highest test substance concentration should produce cytotoxicity (< 90% cell viability) unless5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test substance.

51.30

39.17

PASS

Measurement of CD54 and CD86 Expression

Criterion

Run 1

Run 2

Outcome

Cell viabilities of medium and solvent controls should be higher than 90%

97.07

86.61

PASS

In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) compared to the medium control

N/A

N/A

N/A

For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%

CD54: 159.29%

CD86: 139.77%

CD54: 219.67%

CD86: 179.02%

PASS

In the positive control (Nickel Sulphate), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be greater than 50%.

CD54 RFI: 229

CD86 RFI: 158

CD54 Via:

90.20%

CD86 Via:

90.96%

CD54 RFI: 266

CD86 RFI: 155

CD54 Via:

72.06%

CD86 Via:

67.67%

PASS

For each test substance, the cell viability should be greater than 50% in at least four tested concentrations in each run

7/8

8/8

PASS

Negative results are acceptable only for test substances exhibiting a cell viability of less than 90% at the highest concentration tested, unless 5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test substance.

35.8

90.69

(Test substance is positive)

PASS

RFI - Relative Fluorescence Intensity, MFI - Mean Fluorescence Intensity, DMSO - Dimethyl Sulphoxide

 

c) Results Summary

The CV75 dose informs the dosing range selected for the CD54/86 expression assay. The top dose for the CD54/86 expression assay (main test) is 1.2 x CV75 which was equal to 201.12 µg/mL. The following tables show the expression of CD54 and CD86 against test substance dose with concurrent cytotoxicity measurement:

Run 1 (Valid): Result = Positive

Test Substance Dose (µg/mL)

Cell Viability (%)

Average Cell Viability (%)

CD54 RFI

CD86 RFI

Isotype

CD54

CD86

201.12

30.44

34.90

42.07

35.80

218

160

167.60

45.11

50.45

59.28

51.61

299

206

139.67

50.40

56.94

60.93

56.09

127

108

116.39

54.80

62.05

69.13

61.99

171

123

96.99

62.20

65.90

69.85

65.98

84

74

80.83

54.96

61.37

66.19

60.84

89

98

67.35

62.23

63.06

66.47

63.92

91

108

56.13

58.61

59.35

61.78

59.91

102

74

 

Run 2 (Valid): Result = Positive

Test Substance Dose (µg/mL)

Cell Viability (%)

Average Cell Viability (%)

CD54 RFI

CD86 RFI

Isotype

CD54

CD86

201.12

90.44

90.59

91.05

90.69

1086

1159

167.60

75.29

78.95

75.41

76.55

74

130

139.67

76.50

71.13

76.90

74.84

47

110

116.39

68.33

76.45

74.61

73.13

105

99

96.99

67.61

73.84

74.95

72.13

62

115

80.83

78.53

79.11

72.49

76.71

83

130

67.35

74.22

76.57

77.31

76.03

113

155

56.13

80.29

55.51

80.78

72.19

71

110

 

As can be seen from the data, the expression of CD54 as measured by the RFI crossed the threshold (RFI ≥200) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 µg/mL in Run 2 (highlighted in green). The expression of CD86 as measured by the RFI crossed the threshold (RFI ≥150) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 and 67.35 µg/mL in Run 2 (highlighted in gold). As the CD54/CD86 expression crossed the threshold in both runs the test substance is classified as Positive. Cell viability fell below 50% at the top concentration (201.12 µg/mL) in Run 1 (viability 35.80%, highlighted in pink) however the threshold was also crossed for both markers at 167.60 µg/mL at a non-cytotoxic concentration (51.61%), therefore, the result is deemed to be valid.

 

The EC200 and EC150 values derived for the test substance were as follows:

Marker

EC200 (Rep1)

EC200 (Rep2)

Final EC200 Value

(µg/mL)

CD54

152

172

172

Marker

EC150 (Rep1)

EC150 (Rep2)

Final EC150 Value

(µg/mL)

CD86

152

66

152

 

For test substance the dose that gave 75% cell viability was found to be 167.6 µg/mL. The EC200 and EC150 values for CD54 and CD86 expression were 172 and 152 µg/mL respectively, therefore, Test substance was classified as positive as per the prediction model.

Conclusions:
Under the study conditions, the test substance was concluded to be skin-sensitiser.
Executive summary:

A study was conducted to determine the skin sensitisation potential of test substance, 'Cocodimonium hydroxypropyl hydrolysed wool (62.8% active)', using the in vitro human Cell Line Activation Test (h-CLAT) method, according to OECD 442E, in compliance of GLP. The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1 (a cell line that mimics DCs). The ability of a chemical to induce activation of CD86 and CD54 in THP-1 cells was used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance. The concentration of the test substance used was selected on the basis of a pre-determined CV75 value (i.e. the estimated concentration yielding 75% cell viability). For the CV75 determination, THP-1 cells were pre-cultured for 72 h. Following this, the cells were dosed with the test substance over an 8 dose range (2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 µg/mL) in RPMI culture medium and incubated for 24 ± 0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The concentrations tested for CD54/CD86 expression assay were 201.12, 167.60, 139.67, 116.39, 96.99, 80.83, 67.35 and 56.13 µg/mL. The expression of CD54 as measured by the RFI crossed the threshold (RFI ≥200) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 µg/mL in Run 2. The expression of CD86 as measured by the RFI crossed the threshold (RFI ≥150) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 and 67.35 µg/mL in Run 2. As the CD54/CD86 expression crossed the threshold in both runs the test substance is classified as Positive. Cell viability fell below 50% at the top concentration (201.12 µg/mL) in Run 1 (viability 35.80%) however the threshold was also crossed for both markers at 167.60 µg/mL at a non-cytotoxic concentration (51.61%), therefore, the result was considered to be valid. For test substance the dose that gave 75% cell viability was found to be 167.6 µg/mL. The EC200 and EC150 values for CD54 and CD86 expression were calculated to be 172 and 152 µg/mL respectively; therefore, the test substance was classified as positive as per the prediction model. Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Under the study conditions, the test substance was concluded to be skin-sensitiser (XcellR8, 2018).

Endpoint:
skin sensitisation: in vivo (non-LLNA)
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:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
not specified
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid GMPT study was available before REACH came into force, therefore no additional LLNA study was conducted.
Species:
guinea pig
Details on test animals and environmental conditions:
Number of animals in test group: 20
Number of animals in negative control group: 20

Concentrations of the test substance and vehicle used at each stage of induction
- Intradermal: 0.01% physiol. saline
- Epidermal: 3% physiol. saline
Concentrations of test substance and vehicle used at each stage of challenge:
- Epidermal: 0.01% physiol. saline
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
epidermal: 0.1% in physiological saline
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
very slight erythema
Remarks on result:
other: positive indication only in 10% animals
Remarks:
(not sensitising)
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
epidermal: 0.1% in physiological saline
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
very slight erythema
Remarks on result:
other: positive indication only in 10% animals
Remarks:
(not sensitising)
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
epidermal: 0.1%
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: not sensitising
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
epidermal: 0.1%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested

Signs of irritation during induction: in the test substance group 3/20 animals showed a skin reaction. In the control group 1/20 animals had developed a slight erythema.

Results

 

Number of animals showing skin reaction after

Challenge concentrations of test sub %

1stchallenge

2ndchallenge

24h

48h

24h

48h

Test group 0

2

2

 

 

Negative control group 0

1

0

 

 

Evidence of sensitization at each challenge

Very slight erythema

Interpretation of results:
other: not classified based EU CLP criteria
Conclusions:
Based on results of the read across study, the test substance is considered to be non-sensitising in guinea pigs.
Executive summary:

A study was conducted to determine the skin sensitisation potential of the read across substance, 'Quab 360' (35% aqueous solution) in a guinea pig maximisation test (GPMT), according to OECD Guideline 406. In the study, 20 animals were treated with the test substance and another 20 were treated only with vehicle. The guinea pigs were exposed to 0.01% intradermal applications of the test substance in physiological saline followed by 3% epidermal applications of the test substance in physiological saline during the induction phase. After a rest period of 2 weeks, the animals were challenged with 3% epidermal applications of the test substance in physiological saline. Only 4/20 and 1/20 animals showed very slight erythema reactions in the read across substance and the control groups respectively. No other observations were noted. Under the study conditions, the read across substance was concluded to be non-sensitising in guinea pigs (Degussa, 2007).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

The skin sensitisation potential of the test substance, ‘Cocodimonium hydroxypropyl hydrolysed wool’, has been assessed based on in silico and in vitro sensitisation studies addressing the sequential events, as described in the adverse outcome pathway (AOP 40:1) for skin sensitisation. As per this AOP, the sequence starts with a molecular initiating event (MIE), which is covalent binding of the test substance to the skin proteins, followed by keratinocytes' activation (as key event 1 (KE 1); and activation of dendritic cells (as KE 2). These results are further supported with weight of evidence from studies on substances representative of 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). All results are presented below:

In silico:

Sensitization profiling as per OECD QSAR Toolbox v.4.1

Profilers

Background information

Cocodimonium hydroxypropyl hydrolysed wool

 

Representative structure containing glutamic acid

Representative structure containing arginine

Representative structure containing arginine/glutamic as dipeptide

Protein binding potency Cys (DPRA 13%)

(v.01)

Profiles built in relation with the implementation of the adverse outcome pathway (AOP) forskin sensitization. Developed based on data from the Direct Peptide Reactivity Assay (DPRA).The set of structural alerts of each profile are separated into three potency categories: DPRA above 21% (DPRA 13%), DPRA less than 9% (DPRA 13%) and Grey zone 9-21% (DPRA 13%).

DPRA less than 9% (DPRA 13%) >> Alcohols[1]

DPRA less than 9% (DPRA 13%) >> Cationic surfactants[2]

DPRA less than 9% (DPRA 13%) >> Non-Conjugated carboxylic acids and esters (non-reactive)[3]

Protein binding potency Lys (DPRA 13%)

(v.01)

DPRA less than 9% (DPRA 13%) >> Alcohols[4]

DPRA less than 9% (DPRA 13%) >> Non-Conjugated carboxylic acids and esters (non-reactive)3

DPRA less than 9% (DPRA 13%) >> Alcohols4

DPRA less than 9% (DPRA 13%) >> Guanidine[5]

DPRA less than 9% (DPRA 13%) >> Non-Conjugated carboxylic acids and esters (non-reactive)

Protein binding by OASIS

(v.1.5)

Method particularly relevant forskin and respiratory sensitization, but also forchromosomal aberration.

No alert found

Protein binding by OECD

(v.2.3)

 

No alert found

Protein binding potency

(v.2.4)

 

Not possible to classify according to these rules (GSH)[6]

Keratinocyte gene expression

(v.2.0)

Profile built in relation with the implementation of the adverse outcome pathway (AOP) forskin sensitization. Developed based on data from the KeratinoSens assay.

Not possible to classify according to these rules[7]

Protein binding alerts for skin sensitization by OASIS

(v.1.5)

Alerts developed by industry consortia and LCMas a part of the TIMES model to predict skin sensitisation.The profile focuseson the presence of alerts responsible of the interaction with proteins and especially with skin proteins.

No alert found

Protein binding alerts for skin sensitization according to GHS

(v.1.0)

The profiler is based on recently developed OASIS TIMES model for predicting skin sensitization according to GHS criteria. The protein binding alerts are extracted from 517 chemicals used as a training set for the model.

No alert found

Protein Binding Potency h-CLAT

(v.1.0)

This profile is built in relation with the implementation of the AOP for skin sensitization and developed on the basis of data derived from the human cell line activation (h-CLAT) assay.

No alert found

Respiratory sensitization

(v.1.1)

This profiler is intended to be used for the assessment of respiratory sensitisation potential of low molecular weight chemicals. The profiler is suitable for identifying chemicals capable of inducingrespiratory sensitisationvia both the skin and lung.

No alert found


[1]Short-chain aliphatic alcohols show almost no reactivity (values approximately zero); at the best, some non-covalent interactions such as formation of O-H---S hydrogen bonds may exist. For longer-chain aliphatic alcohols and diols, and benzyl alcohol, these non-covalent interactions are assumed to occur at somewhat higher degree, due to the increase of alkyl chain length and the enhanced electrophilicity of benzylic carbon. No other functionalities with potential covalent reactivity towards cysteine have been identified for this class of compounds.

[2]Surfactants could be considered as false positives in the DPRA test. They do not form stable covalent bonds with the –SH and –NH2 groups of cysteine and lysine peptides, respectively. It could be suggested that the peptide depletion is a result of solubilizing or denaturation of peptides in the presence of surfactants in the test media. This theory is supported by the fact that surfactants are commonly used in protein chemistry as agents assisting in protein/peptide solubilization to denaturating proteins.

[3]Justification under development.

[4]Alkanols, diols and benzyl alcohols show almost no lysine reactivity (values approximately zero as determined experimentally). Non-covalent interactions (formation of hydrogen bonds) exist between the hydroxyl group of alcohols (hydrogen donors) and the primary amines (hydrogen acceptors). By analogy, such interaction could be assumed between alcohols, diols and benzyl alcohols with the primary side amino groups of lysine peptide under the conditions of the DPRA test in buffered aqueous solutions. Such interactions, however, do not elicit appreciable lysine depletion, since no unctionalities capable of reactivity in this respect are present in the molecular structure of the test chemicals.

[5]Guanidine and many of its derivatives such as nitroguanidine, alkylguanidines, alkylnitroguanidines, cyanoguanidine, arylguanidines (1,3-diphenylguanidine, 1,3-di(o-tolyl)guanidine, 1,2,3-triphenylguanidine, etc.), substituted condensation products of guanidine (biguanides, triguanides, etc.),

[6]This profiler is developed based on empirical data for thiol reactivity expressed by thein chemicoRC50 value. Data are obtained by measuring target chemical covalent binding with the thiol group of glutathione (GSH).

[7]The target chemical does not match any of the criteria specified in the profiling scheme.

In vitro study with the test substance:

KeratinoSensTMstudy (KE-1)

An in vitro study was conducted to determine skin sensitisation potential of the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool' (active: 62.8%) using ARE-Nrf2 Luciferase test method, according to OECD Guideline 442D, in compliance with GLP. A single application of 12 concentrations of the test substance (i.e., 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, 0.098, 0.049, 0.024 µg/mL) and 5 concentrations of the positive control (ranging from 8µM to 128 µM) were applied in cell culture medium (dilution factor of 2) with a final concentration of Ethanol of 1%. A single application of culture medium with 1% Ethanol was applied as the negative control. After 24 h, cells were incubated with the test or reference substance for 48 ± 2h at 37C, 5% CO2, ≥ 95% relative humidity before endpoints measurements. Three repetitions were performed and each repetition consisted of 3 x 96-well plates for luminescence and 2x 96-well plates for MTT. The sensitisation potential of the test substance was quantified by calculating two parameters known as the EC1.5 (i.e., effective concentration of test substance that caused an induction of luciferase activity of greater than 1.5-fold over untreated controls) and Imax value (i.e., maximum-fold induction observed within the concentration range tested). The validity of each repetition was assessed following acceptance criteria. In each repetition of the study, no concentration of the test substance caused luciferase induction ≥1.5. Therefore, the test substance was classified as non-sensitising. The maximum induction was observed at a test concentration of 0.391 µg/mL, which showed an Imax value of 0.986. For reference, during test validation, sensitising proficiency chemicals produced Imax values of up to 36-fold over untreated controls. All of the formal acceptance criteria of the tests were met apart from acceptance criterion 2. However, as all other acceptance criteria were met and there was a dose-dependent increase of induction with the positive control, results were considered as valid by the study author. Under the study conditions, the test substance was considered to be non-sensitising to skin (XcellR8, 2017). 

h-CLAT study (KE-2):

A study was conducted to determine the skin sensitisation potential of test substance, 'Cocodimonium hydroxypropyl hydrolysed wool (62.8% active)', using the in vitro human Cell Line Activation Test (h-CLAT) method, according to OECD 442E, in compliance of GLP. The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1 (a cell line that mimics DCs). The ability of a chemical to induce activation of CD86 and CD54 in THP-1 cells was used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance. The concentration of the test substance used was selected on the basis of a pre-determined CV75 value (i.e. the estimated concentration yielding 75% cell viability). For the CV75 determination, THP-1 cells were pre-cultured for 72 h. Following this, the cells were dosed with the test substance over an 8 dose range (2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 µg/mL) in RPMI culture medium and incubated for 24 ± 0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The concentrations tested for CD54/CD86 expression assay were 201.12, 167.60, 139.67, 116.39, 96.99, 80.83, 67.35 and 56.13 µg/mL. The expression of CD54 as measured by the RFI crossed the threshold (RFI ≥200) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 µg/mL in Run 2. The expression of CD86 as measured by the RFI crossed the threshold (RFI ≥150) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 and 67.35 µg/mL in Run 2. As the CD54/CD86 expression crossed the threshold in both runs the test substance is classified as Positive. Cell viability fell below 50% at the top concentration (201.12 µg/mL) in Run 1 (viability 35.80%) however the threshold was also crossed for both markers at 167.60 µg/mL at a non-cytotoxic concentration (51.61%), therefore, the result was considered to be valid. For test substance the dose that gave 75% cell viability was found to be 167.6 µg/mL. The EC200 and EC150 values for CD54 and CD86 expression were calculated to be 172 and 152 µg/mL respectively; therefore, the test substance was classified as positive as per the prediction model. Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Under the study conditions, the test substance was concluded to be skin-sensitiser (XcellR8, 2018).Overall, the weight of evidence from in silico and in vitro read across studies are summarised below:

MIE (covalent binding to proteins):The recommended DPRA assay could not be run as the substance is an UVCB substance. However, the OECD toolbox v. 4.1 profiling (Protein binding potency Cys/Lys (DPRA 13%)) indicated less than 9% binding, i.e. low peptide reactivity. In addition, all the profilers related to the endpoint indicated absence of alert.

KE 1 (Keratinocyte activation):Keratinosens (ARE-Nrf2 Luciferase) assay results were negative for keratinocyte activation.

KE 2 (DC activation):h-CLAT assay result was positive, which indicated DC activation. Test results are considered to be questionable due to lack specificity of current tests to differentiate between immunogenicity and allergenicity for proteins (Basketter and Kimber, 2018), which can be extended for protein derivatives (such as the substance to be registered) as well.

In vivo studies on main reactants:

Hydrolysed proteins

Study 1:A study was conducted to determine the skin sensitisation potential of a read across substance, Hydrolysed keratin (active content not specified) to induce primary or cumulative irritation and/or allergic contact sensitization in repeat insult patch test in humans (HRIPT), in compliance with ICH E6 GCP (Good Clinical Practice) and 21 CFR Part 50 and 56. Fifty eight human volunteers, male and female, ranging in age from 18 to 79 years, were selected for this evaluation (fifty one human volunteers completed this study, while remaining subjects discontinued their participation for various reasons, none of which were related to the application of the read across). Approximately 0.2 mL of the read across, or an amount sufficient to cover the contact surface, was applied to the 1" x 1" absorbent pad portion of a clear adhesive dressing. This was then applied to the upper back between the scapulae to form a semi-occluded patch. Patches were applied three times per week for a total of nine applications. Following supervised removal and scoring of the first Induction patch, participants were instructed to remove all subsequent induction patches at home, 24 h after application. The evaluation of this site was made again just prior to re-application. If a participant was unable to report for an assigned test day, one makeup day was permitted. This day was added to the induction period. With the exception of the first supervision induction patch reading, if any test site exhibited a moderate (2-level) reaction during the induction phase, application was moved to an adjacent area. Applications were discontinued for the remainder of this test phase, if a moderate (2-level) reaction observed on this new test site. Applications were also discontinued if marked (3- level) or severe (4-level) reactivity was noted. Rest periods consist of 24 h following each Tuesday and Thursday removal, and 48 h following each Saturday removal. Approximately 2 weeks after the final induction patch application, a challenge patch was applied to a virgin test site adjacent to the original induction patch site, following the same procedure as induction. The patch was removed and the site scored at the clinic 24 h and 72 h post-application. Based on the grading and evaluations of study results, the study author concluded that read across did not induce dermal irritation or allergic contact sensitization throughout the test interval (CPT, 2004).

Study 2: A study was conducted to determine the skin sensitisation potential of a read across substance, Hydrolysed keratin (active: 2.3%) to induce primary or cumulative irritation and/or allergic contact sensitization in repeat insult patch test in humans (HRIPT). Fifty eight human volunteers, male and female, ranging in age from 17 to 75 years, were selected for this evaluation (fifty seven human volunteers completed this study, while remaining subject discontinued her participation for personal reasons, none of which were related to the application of the read across substance). Prior to the initiation of the study, the read across substance was prepared as a 10% dilution using distilled water. Approximately 0.2 mL of the read across substance, or an amount sufficient to cover the contact surface, was applied to the 1" x 3/4" gauze portion of a clear adhesive dressing. This was then applied to the upper back between the scapulae to form a semi-occluded patch. Patches were applied three times per week for a total of ten applications. The participants were instructed to remove all subsequent induction patches at home, 24 h after application. The evaluation of this site was made again just prior to re-application. If a participant was unable to report for an assigned test day, one makeup day was permitted. This day was added to the induction period. With the exception of the first supervision induction patch reading, if any test site exhibited a moderate (2-level) reaction during the induction phase, application was moved to an adjacent area. Applications were discontinued for the remainder of this test phase, if a moderate (2-level) reaction observed on this new test site. Applications were also discontinued if marked (3- level) or severe (4-level) reactivity was noted. Rest periods consist of 24 h following each Tuesday and Thursday removal, and 48 h following each Saturday removal. Approximately two weeks after the final induction patch application, a challenge patch was applied to the original site and to a virgin test site. The patch was removed and the site scored at the clinic 24 h and 72 h post-application. The volar forearm served as the virgin test site. Based on the grading and evaluations of study results, the study author concluded that read across substance did not induce dermal irritation or allergic contact sensitization throughout the test interval (CPT, 1997).

Expert opinions on hydrolysed proteins:

Cosmetic Ingredient Review (CIR) Expert Panel:A CIR (2014) report on hydrolyzed wheat protein (HWP) and hydrolyzed wheat gluten (HWG), reported a human repeated insult patch test (HRIPT) study with HWP (MW = 350 Da), which was not found to be a dermal irritant during the induction phase or sensitising during the challenge phase of the study. HWG and HWP are known to have safe use history up to 0.01 and 1.7%, respectively in rinse off cosmetic products. Multiple cases of allergic reactions, including Type 1 immediate hypersensitivity reactions, were reported in individuals who had used personal care products that contained HWP, most of which were to a facial soap in Japan that contained HWP of 40,000-50,000 Da from acid hydrolysis of gluten at high temperatures. Several studies were conducted to characterize the cause, manifestations, and mechanisms of these reactions, including tests of serum IgE binding and reactivity to wheat protein, wheat-protein fractions, and HWP and HWG prepared using acid- and/or enzyme-hydrolysis methods yielding products with varied polypeptide size profiles. From these experiments, it was found that hydrolysates with weight-average MWs < 3000 exhibit no potential to elicit hypersensitivity reactions in sensitized individuals, in contrast to hydrolysates with weight-average MWs >30,000 Da. The experimental results supported the hypothesis that polypeptides with weight-average MWs of 3500 Da or less do not have the potency required to induce Type 1 hypersensitivity. The CIR Panel therefore concluded that data from clinical and laboratory studies was sufficient to demonstrate that these ingredients will not elicit Type 1 immediate hypersensitivity reactions in sensitized individuals, and will not induce sensitization when the polypeptide lengths of the hydrolysates do not exceed 30 amino acids. The Panel concluded that HWG and HWP are safe in cosmetics when formulated to restrict peptides to a weight average of 3500 daltons (Da) or less (CIR, 2014).

Scientific Committee on Consumer Safety (SCCS):The SCCS in Europe published a revised opinion on the safety of hydrolysed wheat proteins (HWP) in cosmetic products in 2014. In view of the numbers of reported cases of immediate-type contact urticarial and systemic allergic reactions, the overall risk of sensitization to HWP appears to be low, with the exception of an ‘epidemic’ in Japan associated with one particular HWP product used in some brands of soap. Scientific concerns with regard to the use of HWP in cosmetic products include that there is evidence that sensitisation to HWP is via exposure to cosmetics, not via food. Also there are indications that the risk of sensitisation is higher when HWP’s of higher molecular weight are used on the skin, in particular as an ingredient of products that have strong surfactant properties such as soaps and liquid soaps. The SCCS considered the use of hydrolysed wheat proteins safe for consumers in cosmetic products, provided that the maximum molecular weight average of the peptides in hydrolysates is 3.5 kDa (SCCS, 2014).

Basketter and Kimber:Independent Consultants gave their commentary on minimising the risk of allergy to HWP. The authors reviewed the literature on safe use history and occasional allergic reactions reports. They have also reviewed the CIR and SCCS opinion on the HWPs. They have opined that the draft opinion of the SCCS is based on the Japanese Glupearl experience and thereby seeks to limit the risk of HWP allergy in association with cosmetic products. However, they conclude that the optimal approach is to eliminate the hazard at source by imposing an upper limit of molecular weight as proposed by the CIR. Application of a 3.5kD cap on the molecular weight would effectively remove the hazard, such that HWP could continue to be used safely in all cosmetic products.

In general, the protein hydrolyzates are abundantly available in the environment, in food and extensively distributed throughout the human body, and therefore are not expected to have a sensitisation potential.

Further, the above authors also recently published an article (Basketter and Kimber, 2018), where they raised questions with regard to the specificity of the existing in vitro and in vivo skin sensitisation tests for determination of skin sensitisation potential of chemicals compared to proteins. This is because, unlike chemicals, all proteins can induce an immune response but none of the current in vitro/in vivo tests can make a distinction between immunogenicity (which induces IgG antibody production) and allergenicity (which induces both IgE and IgG antibody production). Therefore, due to lack of this specificity together with absence of suitable body of either positive or negative controls, dictates that use of these tests with proteins is without any scientific justification or predictive merit.

Quaternary ammonium compounds (QAC) – Quab 360

A study was conducted to determine the skin sensitisation potential of the read across substance, ‘Quab 360’ (35% aqueous solution) in a guinea pig maximisation test (GPMT), according to OECD Guideline 406. In the study, 20 animals were treated with the test substance and another 20 were treated only with vehicle. The guinea pigs were exposed to 0.01% intradermal applications of the test substance in physiological saline followed by 3% epidermal applications of the test substance in physiological saline during the induction phase. After a rest period of 2 weeks, the animals were challenged with 3% epidermal applications of the test substance in physiological saline. Only 4/20 and 1/20 animals showed very slight erythema reactions in the read across substance and the control groups respectively. No other observations were noted. Under the study conditions, the read across substance was concluded to be non-sensitising in guinea pigs (Degussa, 2007).

Overall, based on the above weight of evidence from in silico (OECD QSAR Tool box v.4.1 profiling) together with in vitro studies (KeratinoSensTMassays and h-CLAT) together with supportive in vivo evidence from studies on the main reactants, the test substance, ‘Cocodimonium hydroxypropyl hydrolysed wool’ is considered to be non-sensitising to the skin.

References:

1) CIR 2014. Safety assessment of hydrolyzed wheat protein and hydrolyzed wheat gluten as used in cosmetics, June 14, 2014. Available at http://www.cir-safety.org/sites/default/files/wheatp062014final.pdf (accessed in April 2018).

2) SCCS 2014. Opinion on Hydrolysed wheat proteins, SCCS/1534/14 June 18, 2014. Available at ec.europa.eu/health/scientific_committees/consumer_safety/docs/sccs_o_160.pdf (accessed in April 2018).

3) Basketter D, Kimber I 2014. Hydrolysed wheat proteins: A commentary on minimising the risk of allergy, July 2014 (an independent assessment by 3rd Party skin sensitisation experts for Croda).

4) Basketter D, Kimber I 2018. Are skin sensitisation test methods relevant for proteins? Reg. Toxicol. and Pharmacol (article in press): doi: https://doi.org/10.1016/ j.yrtph.2018.09.028.

Justification for classification or non-classification

Based on weight of evidence studies results, the test substance, 'Cocodimonium hydroxypropyl hydrolysed wool' does not warrant classification for skin sensitisation, according to the EU CLP criteria (Regulation 1272/2008/EC).