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Administrative data

Description of key information

Testing data

There is no in vivo study available for this material.

Two in vitro studies have been performed for this substance.

1-     DPRA (Study# 519609, Charles River Laboratories)

FLORANTONE T was negative in this in chemico assay and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

2-     KeratinoSens (Study# 519610, Charles River Laboratories)

There was a slight increase of the luciferase activity after treatment with your test item. In the first experiment was the induction of 1.41-fold increase (just below 1.5 fold) and in the second experiment a 1.98-fold increase (just above the cut-off value). Therefore there was a negative and positive result. In the third experiment, a 1.66-fold increase was observed.

Overall, the test itemis classified as positive in the KeratinoSensTMassay since positive results (>1.5-fold induction) were observed at test concentrations with a cell viability of >70% compared to the vehicle control in two out of three experiments at test concentrations <1000 µM.

Non testing method

According to the OECD Toolbox v4.1

No structural alert has been identified by any of the profiler. In addition, the Protein binding potency Cys/ Lys is negative, which is in line with the in vitro result.

The Keratinosens Profiler returned the following “Not possible to classify” as the substance does not match any of the criteria specified in the profiling scheme.

There was no alert according to the Protein binding Potency h-CLAT.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 September 2017 - 20 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The DPRA assay is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
03 November 2015
Type of study:
direct peptide binding assay
Test material information:
Composition 1
Details on study design:
TEST ITEM PREPERATION
No correction for the purity/composition of the test item was performed.
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), MQ/ACN (1:1, v/v), isopropanol, acetone, acetone/ACN (1:1, v/v) and dimethylsulfoxide (DMSO)/ACN (1:9, v/v).
Test item stock solutions were prepared freshly for each reactivity assay. For the cysteine and lysine reactivity assay, respectively, 28.39 mg and 27.54 mg of test item were pre-weighed into a clean amber glass vial and dissolved, just before use, in 1629 μL and 1581 μL ACN, respectively, to obtain 100 mM solutions. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac- RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL. The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Calibration curve SPCC and SPCL: according to guideline
- Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- and RClysB-sample were 24.5 hours and 23 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.
- Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1 ('Other information on methods and materials'). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 ('Other information on materials and methods').

POSITIVE CONTROL: Cinnamic aldehyde
- Purity: 98.4%

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration, and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]*100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample a ratio in the range of 90%< mean area ratio of control samples <110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION (see also 'Other information on materials and method')
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
Positive control results:
The positive control had a mean SPCC depletion of 75.4 ± 2.5% and a mean SPCL depletion of 49.2 ± 0.8%.
Key result
Parameter:
other: SPCC mean depletion (%)
Run / experiment:
Cysteine Reactivity Assay
Value:
0.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Remarks:
SD: 1.6%
Key result
Parameter:
other: SPCL mean depletion (%)
Run / experiment:
Lysine Reactivity Assay
Value:
0
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Since precipitation was observed upon addition of the test item to the SPCL peptide solution, one cannot be sure how much test item remained in the solution to react with the SPCL peptide. The negative result is therefore uncertain and should be interpreted with due care.

In both the cysteine reactivity assay and the lysine reactivity assay, all acceptability criteria were met and the assays are considered valid (see table 4).

Table 4 Acceptability of the DPRA assay

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

>0.99

0.994

>0.99

0.995

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.505 ± 0.007

0.50 ± 0.05

0.504 ± 0.004

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

 0.494 ± 0.009

0.50 ± 0.05

0.494 ± 0.005

CV (%) for RC samples B and C

<15.0

2.0

<15.0

2.0

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

75.4

40.2-69.0

49.2

SD of peptide depletion cinnamic aldehyde (%)

<14.9

2.5

<11.6

0.8

SD of peptide depletion for Florantone T (%)

<14.9

1.6

<11.6

0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation; NA = Not Applicable.

* For calculation of the RC-C mean and the CV, the RCcysC-3 value was excluded (outlier due to injection error). As a result, no SD could be calculated for the mean of the RCcysC.

Table 5 SPCC and SPCL depletion and reactivity classification for Florantone T

Test item

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

Reactivity class

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

Florantone T

0.9%

±1.6%

0.0%

±0.0%

0.5%

Negative: No or minimal reactivity

Interpretation of results:
study cannot be used for classification
Remarks:
Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
Florantone T was negative in a DPRA performed according to OECD guideline 442C and GLP principles. The test item was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. The study cannot be used stand-alone to draw a conclusion on classification.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 September 2017 - 06 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd. 3 November 2015
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The purpose of this study was to evaluate the ability of Florantone T to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. Activation of this pathway can lead to skin sensitisation.
Test material information:
Composition 1
Specific details on test material used for the study:
Specific gravity/density: 1.059 (20ºC); 1.057 (25ºC)
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:

Number of replicates: three independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

CONTROLS:
Positive control: ethylene dimethacrylate glycol (purity: 98.3%), 7.8-250 µM, tested in triplicate, DMSO was used as a vehicle;
Negative control: DMSO (1% in exposure medium);
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values.

TEST SYSTEM:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was 20 in experiment 1, 22 in experiment 2 and 24 in experiment 3.

TEST ITEM PREPARATION:
The test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM (clear colorless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.

TEST CONCENTRATIONS:
- Both experiments: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM

MEDIA:
Basic medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/ml).
Exposure medium
Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

TREATMENT OF CELLS:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2. Initially, experiment 1 did not pass all the acceptability criteria and therefore this part of the study was repeated. In total 3 valid experiments were performed.

LUCIFERASE ACTIVITY MEASUREMENT:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time two seconds).

CYTOTOXICITY ASSESSMENT:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
Positive control results:
The EC1.5 of the positive control was between 5 and 125 μM (59 μM and 78 μM in experiment 1 and 2, respectively). A dose related response was observed and the induction at 250 μM was higher than 2-fold (2.27-fold and 2.18-fold in experiment 1 and 2, respectively).
Key result
Parameter:
other: Imax
Run / experiment:
1
Value:
1.41
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Parameter:
other: Imax
Run / experiment:
2
Value:
1.98
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Parameter:
other: Imax
Run / experiment:
3
Value:
1.66
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Other effects / acceptance of results:
- In the first experiment, the test item showed toxicity (IC30 value of 613 μM, IC50 value of 724 μM). No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.41 and therefore no EC1.5 could be calculated.
- In the second experiment, the test item showed toxicity (IC30 value of 327 μM, IC50 value of 376 μM). A dose related luminescence activity induction was observed after treatment with the test item. The Imax was 1.98 and the EC1.5 134 μM.
- In the third experiment, the test item showed toxicity (IC30 value of 591 μM, IC50 value of 708 μM)

- In both experiments, no precipitation was observed at the start and end of the incubation period in the 96-well plates.

All three experiments passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (88 μM, 35 μM and 20 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.14-fold, 3.74-fold and 5.37-fold in experiment 1, 2 and 3, respectively).
- The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (18.5%, 17.9% and 6.0% in experiment 1, 2 and 3, respectively).

Table1          Overview Luminescence Induction and Cell Viability of the test item in Experiment 1 and 2

Concentration (µM)

0.98

2.0

3.9

7.8

16

31

63

125

250

500

1000

2000

Exp 1 luminescence

1.00

0.97

1.02

1.01

1.03

1.14

0.96

0.91

1.26

1.41

0.00

0.00

Exp 1 viability (%)

114.2

103.0

101.5

99.3

97.5

97.9

93.7

104.4

81.8

90.5

0.1

1.3

Exp 2 luminescence

0.62

0.60

0.65

0.73

0.82

0.88

1.08

1.46

1.98***

0.18

0.00

0.00

Exp 2 viability (%)

102.9

99.2

95.3

96.8

91.2

89.5

90.8

97.7

100.9

0.1

1.2

2.1

Exp 3 luminescence

1.09

1.11

1.14

1.09

1.10

1.16

1.18

1.32

1.45

1.66***

0.00

0.00

Exp 3 viability (%)

100.7

98.9

93.6

92.2

93.0

92.9

85.1

87.1

84.6

85.6

0.2

0.4

***p<0.001 Student’s t test 

Table 2 Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)

7.8

16

31

63

125

250

Exp 1 luminescence

1.03

1.13

1.20

1.28

1.81***

2.14***

Exp 1 viability (%)

106.5

111.9

114.5

119.5

136.6

123.8

Exp 2 luminescence

1.07

1.16

1.44

1.93***

2.90***

3.74***

Exp 2 viability (%)

100.8

104.4

100.5

114.9

110.4

115.1

Exp 3 luminescence

1.22

1.47

1.59*

2.07**

2.43***

5.37***

Exp 3 viability (%)

97.6

98.6

101.6

104.5

110.4

119.7

***p<0.001 Student’s t test

 

Table 3 Overview EC1.5, Imax, IC30 and IC50Values

 

EC1.5(µM)

Imax

IC30(µM)

IC50(µM)

Test item Experiment 1

NA

1.41

613

724

Test item Experiment 2

134

1.98

327

376

Test item Experiment 3

311

1.66

591

708

Pos Control Experiment 1

88

2.14

NA

NA

Pos Control Experiment 2

35

3.74

NA

NA

Pos Control Experiment 3

20

5.37

NA

NA

NA = Not applicable


 

 

Interpretation of results:
study cannot be used for classification
Remarks:
This study is part of a weight of evidence approach on which the classification is based.
Conclusions:
Based on the outcome of a KeratinoSens™ assay performed according to OECD guideline and GLP principles, Florantone T is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Endpoint:
skin sensitisation, other
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with limited documentation / justification
Conclusions:
No structural alert has been identified by any of the profiler. In addition, the Protein binding potency Cys/ Lys is negative, which is in line with the in vitro result.
The Keratinosens Profiler returned the following “Not possible to classify” as the substance does not match any of the criteria specified in the profiling scheme.
There was no alert according to the Protein binding Potency h-CLAT.
Executive summary:

There is no structural alert with the OECE Toolbox v4.1.

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

Justification for classification or non-classification