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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 February 2018 to 22 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 442E: hCLAT human Cell Line Activation Test
Version / remarks:
(2017)
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay was performed by XTT test instead of flow cytometry.
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
The Human Cell Line Activation Test (h-CLAT) was conducted as an alternative to in vivo skin sensitisation tests. The selected in vitro skin sensitisation test was considered scientifically valid for the evaluation of skin sensitisation hazard of chemicals, based on the ECVAM. Information from this test should be used in combination with other information within a weight-of-evidence approach and not as a stand-alone test method.

Test material

Constituent 1
Reference substance name:
D,L- Menthol / D,L-Isomenthol
Molecular formula:
C10H20O
IUPAC Name:
D,L- Menthol / D,L-Isomenthol

In vitro test system

Details on the study design:
DETAILS ON TEST SYSTEM
- Species: Human monocytic leukaemia cell line
- Method(s): XTT test and h-CLAT test
- Expiration date: Not specified
- Date of initiation of testing: 09 October 2019

TEMPERATURE USED FOR TEST SYSTEM
Temperature used during treatment/exposure: The assays were incubated at 37°C
Incubation duration: 24 ± 0.5 hours

CONTROL SAMPLES
Controls: A culture medium with no additives was used as the blank and vehicle control; DMSO and DNCB was used as a positive control.

AMOUNT/CONCENTRATION APPLIED
Test items concentrations: 17, 21, 25, 30, 36, 43, 52 and 62 μg/mL
Positive control concentrations: The concentration for the XTT test were 2.0 and 3.0 μg/mL
Replicates: 7 replicates were used for each test item concentration

Results and discussion

Positive control results:
The actual RFI values for CD86 were 519.2 %, 674.2%, 725.3% and 652.8% and 230.8 %, 482.7%, 296.9% and 342.0% for CD54. The RFI values of both CD86 and CD54 antibodies fulfilled the positive control criteria (CD86 ≥ 150% and CD54 ≥ 200%).

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1/ Experiment 1: CD 54 (17 μg/mL)
Parameter:
other: RFI (%)
Value:
95.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1/ Experiment 1: CD 86 (17 μg/mL)
Parameter:
other: RFI (%)
Value:
98.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2/ Experiment 1: CD 54 (21 μg/mL)
Parameter:
other: RFI (%)
Value:
109.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2/ Experiment 1: CD86 (21 μg/mL)
Parameter:
other: RFI (%)
Value:
96.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3/ Experiment 1: CD 54 (25 μg/mL)
Parameter:
other: RFI (%)
Value:
117.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3/ Experiment 1: CD 86 (25 μg/mL)
Parameter:
other: RFI (%)
Value:
87.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 4/ Experiment 1: CD 54 (30 μg/mL)
Parameter:
other: RFI (%)
Value:
87
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 4/ Experiment 1: CD 86 (30 μg/mL)
Parameter:
other: RFI (%)
Value:
64.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 5/ Experiment 1: CD 54 (36 μg/mL)
Parameter:
other: RFI (%)
Value:
93.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 5/ Experiment 1: CD 86 (36 μg/mL)
Parameter:
other: RFI (%)
Value:
68.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 6/ Experiment 1: CD 54 (43 μg/mL)
Parameter:
other: RFI (%)
Value:
63
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 6/ Experiment 1: CD 86 (43 μg/mL)
Parameter:
other: RFI (%)
Value:
64.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 7/ Experiment 1: CD 54 (52 μg/mL)
Parameter:
other: RFI (%)
Value:
90.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 7/ Experiment 1: CD 86 (52 μg/mL)
Parameter:
other: RFI (%)
Value:
81.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 8/ Experiment 1: CD 54 (62 μg/mL)
Parameter:
other: RFI (%)
Value:
79.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 8/ Experiment 1: CD 86 (62 μg/mL)
Parameter:
other: RFI (%)
Value:
75.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1/ Experiment 2: CD 54 (17 μg/mL)
Parameter:
other: RFI (%)
Value:
119.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1/ Experiment 2: CD 86 (17 μg/mL)
Parameter:
other: RFI (%)
Value:
84.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2/ Experiment 2: CD 54 (21 μg/mL)
Parameter:
other: RFI (%)
Value:
111.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2/ Experiment 2: CD 86 (21 μg/mL)
Parameter:
other: RFI (%)
Value:
105.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3/ Experiment 2: CD 54 (25 μg/mL)
Parameter:
other: RFI (%)
Value:
101.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3/ Experiment 2: CD 86 (25 μg/mL)
Parameter:
other: RFI (%)
Value:
101.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 4/ Experiment 2: CD 54 (30 μg/mL)
Parameter:
other: RFI (%)
Value:
91.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 4/ Experiment 2: CD 86 (30 μg/mL)
Parameter:
other: RFI (%)
Value:
70.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 5/ Experiment 2: CD 54 (36 μg/mL)
Parameter:
other: RFI (%)
Value:
96
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 5/ Experiment 2: CD 86 (36 μg/mL)
Parameter:
other: RFI (%)
Value:
84.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 6/ Experiment 2: CD 54 (43 μg/mL)
Parameter:
other: RFI (%)
Value:
100.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 6/ Experiment 2: CD 86 (43 μg/mL)
Parameter:
other: RFI (%)
Value:
77
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 7/ Experiment 2: CD 54 (52 μg/mL)
Parameter:
other: RFI (%)
Value:
98.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 7/ Experiment 2: CD 86 (52 μg/mL)
Parameter:
other: RFI (%)
Value:
80.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 8/ Experiment 2: CD 54 (62 μg/mL)
Parameter:
other: RFI (%)
Value:
105.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 8/ Experiment 2: CD 86 (62 μg/mL)
Parameter:
other: RFI (%)
Value:
94.2
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:
- Acceptance criteria met for negative control: The negative control was more than 90 % in comparison to the medium control (100 %).
- Acceptance criteria met for positive control: In the positive control DNCB, the RFI values were 230.8%, 482.7%, 296.9% and 342.0% for CD54 and 519.2%, 674.2%, 725.3% and 652.8% for CD86. All values were within the historical laboratory data (CD54 ≥ 200% and CD86 ≥ 150%). The cell viability was also more than 50 % (73.3%, 70.1% 74% and 73.2%). Therefore, the acceptance criteria for the positive control was fulfilled.
- Acceptance criteria met for variability between replicate measurements: Not specified
- Range of historical values if different from the ones specified in the test guideline: No applicable


Any other information on results incl. tables

Table 1- Results from h-CLAT experiment 1

 

Concentration (µg/mL)

RFI (%) CD 54 Antibody

RFI (%) CD 86Antibody

Cell Viability

(%)

Medium Control

-

100.0

100.0

100.0

DMSO control

-

100.0

100.0

100.0

Positive control (DNCB)

2.0

230.8

519.2

73.3

3.0

482.7

674.2

70.1

 

 

 

Test item

17

95.7

98.6

93.7

21

109.8

96.8

94.1

25

117.4

87.5

94.4

30

87.0

64.8

92.3

36

93.5

68.7

90.3

43

63.0

64.8

95.6

52

90.2

81.5

92.0

62

79.3

75.4

86.3

 

Table 2- Results from h-CLAT experiment 2

 

Concentration (µg/mL)

RFI (%) CD 54 Antibody

RFI (%) CD 86Antibody

Cell Viability

(%)

Medium Control

-

100.0

100.0

100.0

DMSO control

-

100.0

100.0

100.0

Positive control (DNCB)

2.0

296.9

725.3

74.0

3.0

342.0

652.8

73.2

 

 

 

Test item

17

119.4

84.8

98.1

21

111.3

105.8

99.5

25

101.6

101.6

97.2

30

91.1

70.2

92.6

36

96.0

84.1

92.0

43

100.8

77.0

86.6

52

98.4

80.9

85.1

62

105.6

94.2

81.7

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The test item with a log Pow of 3.02 and 3.32, did not cause THP-1 cell activation up to the highest tested concentration (62 μg/mL) under test conditions. Therefore, the test item is considered negative for the third key event of skin sensitisation adverse outcome pathway (AOP).
Executive summary:

The study is assigned a reliability score of 1 (reliable without restrictions) as it followed OECD Guideline 442E: In Vitro Skin Sensitisation: Human cell line activation test (h-CLAT) and is compliant with GLP.

The skin sensitising potential of the test item was determined by the third key event of the skin sensitisation Adverse Outcome Pathway (AOP). The test item concentrations of 17, 21, 25, 30, 36, 43, 52 and 62 μg/mL were used in the definitive study. Two independent runs were conducted for the study.

The negative control was more than 90 % in comparison to the medium control (100 %). In the positive control DNCB, the RFI values were 230.8%, 482.7%, 296.9% and 342.0% for CD54 and 519.2%, 674.2%, 725.3% and 652.8% for CD86. All values were within the historical laboratory data (CD54 ≥ 200% and CD86 ≥ 150%). The cell viability was also more than 50 % (73.3%, 70.1% 74% and 73.2%). Therefore, all validity criteria were met. The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay was performed by XTT test instead of flow cytometry.

The test item with a log Pow of 3.02 and 3.32, did not cause THP-1 cell activation up to the highest tested concentration (62 μg/mL) under test conditions. Therefore, the test item is considered negative for the third key event of skin sensitisation adverse outcome pathway (AOP).