Registration Dossier

Administrative data

Description of key information

sensitising: positive in an in vitro testing battery (protein reactivity (DPRA), activation of keratinocytes (LuSens), activation of dendritic cells (MUSST)); RL2; non-GLP

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report which meets basic scientific principles.
Qualifier:
no guideline available
Principles of method if other than guideline:
In the Direct Peptide Reactivity Assay (DPRA) the reactivity of a test item towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated.
GLP compliance:
not specified
Remarks:
The non-GLP study is used in a WoE approach: this study isadquate for C&L and risk assessment; key parameters are adequately and reliably covered; and adequate and reliable documentation of the study is provided.
Type of study:
direct peptide binding assay
Details on study design:
In the Direct Peptide Reactivity Assay (DPRA) the reactivity of a test item towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated. The test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.
The test substance was solved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide.
The peptide depletion of test-substance incubated samples was compared to the peptide depletion of the NC samples and expressed as relative peptide depletion. For the test substance the mean peptide depletion as average of C- and K-peptide depletion is calculated and used for evaluation of the chemical reactivity.
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA) prepared as a 50 mM solution in acetonitrile
Parameter:
other: lysine peptide depletion
Value:
10.7
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: cystein peptide depletion
Value:
49.3
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

substance

Cysteine-Peptide

Lysine-Peptide

mean of both

depletions [%]

Mean depletion

[%]

SD

Mean depletion

[%]

SD

PC: EGDMA

50.3

4.4

11.9

1.2

31.1

Test substance

49.3

4.0

10.7

0.9

30.0

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that THFMA shows a moderate chemical reactivity in the Direct Peptide Reactivity Assay under the test conditions chosen and may be considered as moderate sensitiser.
Executive summary:

In an in vitro Direct Peptide Reactivity Assay the skin sensitisation potential of THFMA was assessed applying the prediction model proposed in Gerberick et. al (2007). Chemical reactivity has been shown to be well associated with allergenic potency. The reactivity of a test item towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated. Ethylene glycol dimethacrylate was used as positive control.

The mean peptide depletion of THFMA was 30.0% (associated with a moderately reactive test substance), which was in the range of the positive control (31.1%).

According to the classification tree model described by Gerberick et al. highly reactive test substance (mean peptide depletion > 42.47 %) is predicted to be a strong sensitiser, a moderately reactive test substance (22.62 % < mean peptide depletion < 42.47 %) a moderate sensitiser, a test substance of low reactivity (6.38 % < mean peptide depletion < 22.62 %) a weak sensitiser, and a test substance of minimal reactivity (mean peptide depletion < 6.38 %) a non-sensitiser.

 

It can be concluded that THFMA shows a moderate chemical reactivity in the Direct Peptide Reactivity Assay under the test conditions chosen and may be considered as moderate sensitiser.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report which meets basic scientific principles.
Qualifier:
no guideline available
Principles of method if other than guideline:
LuSens assay (LuSens, Bauch et al. 2012). The endpoint measurement is the upregulation of the luciferase activity after 48 hours incubation with test substances. This upregulation is an indicatar for the activation of the Keap1/Nrf2/ARE signaling pathway (Ade et al. 2009, Na/sch 2012, Na/sch & Ernter 2008, Vandebriel et al. 2010).
GLP compliance:
no
Remarks:
The non-GLP study is used in a WoE approach: this study isadquate for C&L and risk assessment; key parameters are adequately and reliably covered; and adequate and reliable documentation of the study is provided.
Type of study:
activation of keratinocytes
Details on study design:
The LuSens assay uses a luciferase reporter cell line (LuSens cells) based on the activation of the antioxidant response element that can be used to assess the keratinocyte activating potential of a substance. The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using the genetically modified keratinocytes (LuSens, Bauch et al. 2012). It employs the reporter gene for luciferase under the contral of an ARE and hence monitors Nrf-2 transcription factor activity. The endpoint measurement is the upregulation of the luciferase activity after 48 hours incubation with test substances. This upregulation is an indicatar for the activation of the Keap1/Nrf2/ARE signaling pathway (Ade et al. 2009, Na/sch 2012, Na/sch & Ernter 2008, Vandebriel et al. 2010).

The cell line LuSens was treated with 6 test substance concentrations for 48 hours in at least two independent experiments with each 3 replicates. Cells were lysed and luciferase induction was evaluated by measuring luminescence signal after substrate addition.
In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A test substance was considered to have an ARE induction potential if the fold induction of luciferase activity was >1.5 and viability determined in the MTT assay was >70% at any test concentration.

Concentrations:
128.38, 154.05, 184.86, 221.83, 266.20, 319.44, 383.33 µg/mL in DMSO

Controls:
The strong sensitizer ethylene glycol dimethacrylate (EGDMA 18 µg /mL) was used as positive contraI and DL-lactic acid (LA. 450 mg/m L) as non-sensitizing negative control.

Parameter:
other: fold induction
Value:
> 1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Luciferase activity after test substance treatment exceeded 1.5 fold induction with respect to the vehicle control at concentrations that did not reduce cell viability below 70% in two independent experiments

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (<1fold induction; viability >70%)
- Acceptance criteria met for positive control: yes (7.17-7.46 fold induction; viability >70%)

Preliminary cytotoxicity assessment

Concentration [µg/mL]

mean viability of 3 replicates

rel. viability [%]

Vehicle control

0.539

100.00

0.5

0.530

98.39

1.0

0.553

102.66

5.0

0.547

101.49

10.0

0.559

103.84

50.0

0.591

109.78

100.0

0.557

103.47

500.0

0.188

34.95

1000.0

0.003

0.54

2000.0

0.001

0.23

 

Main Experiments

Concentration [µg/mL]

1st experiment

2nd experiment

fold induction

rel. viability [%]

fold induction

rel. viability [%]

Vehicle control

1

100

1

100

128.38

n.d.

n.d.

25.41

116.0

154.05

25.29

100.2

29.79

103.2

184.86

28.49

89.5

37.30

97.6

221.83

33.70

82.8

41.86

98.2

266.20

39.89

60.0

48.35

89.7

319.44

37.19

55.4

65.03

65.6

383.33

42.50

39.8

n.d.

n.d.

EGDMA

7.17

111.1

7.46

117.2

LA

0.98

100.5

0.91

103.6

 

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
After 48 hours of exposure to THFMA luciferase activity in LuSens cells was induced. It has to be concluded that THFMA has a keratinocyte activating potential.
Executive summary:

In an in vitro LuSens assay the skin sensitisation potential of THFMA was assessed. The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using genetically modified keratinocytes (LuSens, Bauch et al. 2012). It employs the reporter gene for luciferase under the contral of an ARE and hence monitors Nrf-2 transcription factor activity. The endpoint measurement is the upregulation of the luciferase activity after 48 hours incubation with test substances. This upregulation is an indicatar for the activation of the Keap1/Nrf2/ARE signaling pathway (Ade et al. 2009, Na/sch 2012, Na/sch & Ernter 2008, Vandebriel et al. 2010).

Luciferase activity after treatment exceeded 1.5 fold induction with respect to the vehicle control at concentrations that did not reduce cell viability below 70% in two independent experiments.

After 48 hours of exposure to THFMA luciferase activity in LuSens cells was induced. It has to be concluded that THFMA has a keratinocyte activating potential.

 

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report which meets basic scientific principles.
Qualifier:
no guideline available
Principles of method if other than guideline:
The myeloid U937 skin sensitization test is a dendritic cell activation test to predict skin sensitizing potential. The test is performed using the human pro-monocytic cell line U937 as surrogate for dendritic cells. As readout, the change in the expression of the cell membrane marker CD86 measured by flow cytometry after 48 hours of test substance exposure is determined.
GLP compliance:
no
Remarks:
The non-GLP study is used in a WoE approach: this study is adquate for C&L and risk assessment; key parameters are adequately and reliably covered; and adequate and reliable documentation of the study is provided.
Type of study:
activation of dendritic cells
Details on study design:
Controls
The strong sensitizer ethylenediamine (EDA,70 µg/mL) was used as positive and lactic acid (LA, 200 µg/mL) as non-sensitizing negative control.
 
Cytotoxicity
The cytotoxicity of the test substance was evaluated by flow cytometry using propidium iodide staining after 48hours exposure. For the purpose the CV75 value (estimated concentration that affords 75% cell viability) was derived from the concentration response curve.
In the main test, the test substance was used at five final concentrations determined with regard to the CV75 value: CV75 x 2, CV75, CV75 / 2, CV75 / 4, CV75 / 8.
 
Surface marker expression
After 48 hoursof exposure U937 cells were stained with FITC labeled anti-human-CD86 antibody and propidium iodide, and the fluorescence intensity was analyzed using flow cytometry.
A test substance was predicted to have a dendritic cell activating potential, when the marker expression exceeded the threshold of 1.2 with respect to vehicle treated cells (VC) at any tested sufficiently non-cytotoxic (cell viability >/= 70%) concentration in two independent experiments.

concentrations:
79.18, 158 .36, 316.72, 633.44, 1266.87 µg/mL in culture medium
Positive control substance(s):
yes
Remarks:
ethylene diamine
Parameter:
other: fold CD 86 induction
Value:
>= 1.2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
After 48 hours of exposure to THFMA, CD86 expression was induced in U937 cells at concentrations affording at least 70% viability

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (induction <1.2 fold; viability >70%)
- Acceptance criteria met for positive control: yes (induction 2.21-2.32 fold; viability >70%)

Preliminary cytotoxicity assessment

Concentration

µg/mL

%PI negative cells

Replicate 1

%PI negative cells

Replicate 2

%PI negative cells

Mean

rel.Viability mean

Vehicle control

98.48

99.37

98.925

100.00

0.5

99.48

99.49

99.49

100.57

1

99.31

99.43

99.37

100.45

5

99.30

99.47

99.39

100.46

10

99.41

99.38

99.40

100.48

50

99.21

99.36

99.29

100.36

100

99.00

98.74

98.87

99.94

500

94.39

94.83

94.61

95.64

1000

8.52

27.69

18.11

18.30

2000

n.d.

n.d.

n.d.

n.d.

n.d - no viable cells detected

 

Main Experiments

Concentration [µg/mL]

1st experiment

2nd experiment

CD 86 induction

rel. viability [%]

CD 86 induction

rel. viability [%]

Vehicle control

1.00

100

1.00

100

79.18

1.18*

99.8*

1.46

99.0

158 .36

1.63

98.9

1.87

92.5

316.72

1.74

92.6

2.29

69.4

633.44

1.87

71.0

0.60

24.4

1266.87

2.89

23.7

**

**

LA, 200µg/mL

0.96

99.9

1.11

99.8

EDA, 70 µg /mL

2.21

95.4

2.32

93.9

*=value of only one sample

**= no viable cells detected

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
After 48 hours of exposure to THFMA, CD86 expression was induced in U937 cells at concentrations affording at least 70% viability. From this it has to be concluded that THFMA does induce dendritic cell activation.
Executive summary:

The skin sensitisation potential of THFMA was assessed in an in vitro dendritic cell activation test (Myeloid U937 Skin Sensitization Test (MUSST)). The human pro-monocytic cell line U937 as surrogate for dendritic cells. As readout, the change in the expression of the cell membrane marker CD86 measured by flow cytometry after 48 hours of test substance exposure is determined.

The strong sensitizer ethylene diamine (EDA, 70 µg/mL) was used as positive and lactic acid (LA, 200 µg/mL) as non-sensitizing negative control.

The test substance was tested in a concentration range of 79.18 to 1266.87 µg/mL. Cell viability was decreased below 70% at 1266.87 µg/mL (experiment 1) and 316.72 µg/mL (experiment 2). In experiments 1 and 2 an induction of the expression of CD86 was observed at sufficiently non-cytotoxic concentrations.

After 48 hours of exposure to THFMA, CD86 expression was induced in U937 cells at concentrations affording at least 70% viability. From this it has to be concluded, that THFMA induced dendritic cell activation.

 

 

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Reliable (RL=2), relevant and adequate studies are available to assess the sensitisation potential of THFMA.

 

In vitro tests

In an in vitro Direct Peptide Reactivity Assay the skin sensitisation potential of THFMA was assessed applying the prediction model proposed in Gerberick et. al (2007). Chemical reactivity has been shown to be well associated with allergenic potency. The reactivity of a test item towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated.Ethylene glycol dimethacrylate was used as positive control.

The mean peptide depletion of THFMA was 30.0% (associated with a moderately reactive test substance), which was in the range of the positive control (31.1%).

According to the classification tree model described by Gerberick et al. highly reactive test substance (mean peptide depletion > 42.47 %) is predicted to be a strong sensitiser, a moderately reactive test substance (22.62 % < mean peptide depletion < 42.47 %) a moderate sensitiser, a test substance of low reactivity (6.38 % < mean peptide depletion < 22.62 %) a weak sensitiser, and a test substance of minimal reactivity (mean peptide depletion < 6.38 %) a non-sensitiser.

It can be concluded that THFMA shows a moderate chemical reactivity in the Direct Peptide Reactivity Assay under the test conditions chosen and may be considered as moderate sensitiser.

 

In an in vitro LuSens assay the skin sensitisation potential of THFMA was assessed. The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using genetically modified keratinocytes (LuSens, Bauch et al. 2012). It employs the reporter gene for luciferase under the contral of an ARE and hence monitors Nrf-2 transcription factor activity. The endpoint measurement is the upregulation of the luciferase activity after 48 hours incubation with test substances. This upregulation is an indicatar for the activation of the Keap1/Nrf2/ARE signaling pathway (Ade et al. 2009, Na/sch 2012, Na/sch & Ernter 2008, Vandebriel et al. 2010).

Luciferase activity after treatment exceeded 1.5 fold induction with respect to the vehicle control at concentrations that did not reduce cell viability below 70% in two independent experiments.

After 48 hours of exposure to THFMA luciferase activity in LuSens cells was induced. It has to be concluded that THFMA has a keratinocyte activating potential.

 

The skin sensitisation potential of THFMA was assessed in an in vitro dendritic cell activation test (Myeloid U937 Skin Sensitization Test (MUSST)). The human pro-monocytic cell line U937 as surrogate for dendritic cells. As readout, the change in the expression of the cell membrane marker CD86 measured by flow cytometry after 48 hours of test substance exposure is determined.

The strong sensitizer ethylene diamine (EDA, 70 µg/mL) was used as positive and lactic acid (LA, 200 µg/mL) as non-sensitizing negative control.

The test substance was tested in a concentration range of 79.18 to 1266.87 µg/mL. Cell viability was decreased below 70% at 1266.87 µg/mL (experiment 1) and 316.72 µg/mL (experiment 2). In experiments 1 and 2 an induction of the expression of CD86 was observed at sufficiently non-cytotoxic concentrations.

After 48 hours of exposure to THFMA, CD86 expression was induced in U937 cells at concentrations affording at least 70% viability. From this it has to be concluded, that THFMA induced dendritic cell activation.

 

Evaluation criteria for Individual tests and test results

 

Test Method

Evaluation criteria

Test Result

Test evaluation

Direct Peptide Reactivity Assay (DPRA)

Positive if >/= 6.38%mean peptide depletion

 

Negative if <6.38%mean peptide depletion

30.0% mean peptide depletion

(49.3% cysteine peptide depletion; 10.7% lysine peptide depletion).

Positive

Keratinocyte Activation Assay LuSens

Positive if >/= 1.5 -fold luciferase

activity when viability is >70% of the vehicle control

 

Negative if<1.5-fold luciferase

activity

In at least two independent

experiments ARE-dependent luciferase activity induction above1.5-fold at test substance concentrations that did not reduce cell viability below 70% was observed.

Positive

Dendritic Cell Line Activation Assay

MyeloidU937 SkinSensitization Test (MUSST)

Positive if >/= 1.2-foldof CD86

when viability is >70% of the control

 

Negative if <1.2-fold of CD86

In at least two independent

experiments an induct ion of the expression of CD 86 above 1.2 -fold was observed at sufficiently noncytotoxic

concentration.

positive

 

Based on theseresults, THFMA is predicted to be a skin sensitizer.

 

In vivo testing

In accordance with REACh Regulation, Annex XI, 1.4. in vivo testing on sensitisation is not required for THFMA. For the assessment of the sensitizing potential of the substance, a testing battery of three in vitro tests is available, all three suggesting sensitizing properties:

-         protein reactivity (DPRA),

-         activation of keratinocytes (LuSens),

-         activation of dendritic cells (MUSST)

 

Robust study summaries are included in the dossier.

 

The individual studies of the test battery were performed according to the methods described in the publications cited in the individual study reports. Formal validation studies have been completed for the direct peptide reactivity assay (DPRA), dendritic cell line activation assay h-CLAT and the ARE-dependent keratinocyte activation assay KeratinoSens and initiated for the dendritic cell line activation test MUSST and the ARE-dependent keratinocyte activation assay LuSens.

The combination of test methods and the evaluation of their results has been evaluated and published by Bauch et al.,2012. Based on the performance standards of the OECD test guideline no.429 (Local Lymph Node Assay, LLNA, OECD 2010), the evaluation based on the DPRA, LuSens and MUSST methods yields an overall accuracy of 95% compared to results in humans (for comparison: for the same data set the LLNA yielded an overall accuracy of 86%).

 

In the test battery evaluation a weight of evidence approach is used: Any two of the three tests determine the overall results, i.e., any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer.

 

Ass all three individual studies were positive, THFMA should be considered as a sensitizer. No confirmatory in vivo test is required.

 

Human data

Supporting human data are available:

 

Patch testing of patients with a history of exposure to (meth)acrylates resulted in 5/147 (3.4%) positive cases for THFMA.

 

Clinical records of patients with known occupational allergic contact dermatitis from methacrylates in glues were analysed: Positive reactions to THFMA in the patch test were detected in 7 patients with wide methacrylate allergy (7–10 reactions to different methacrylates), while the 3 THFMA-negative patients reacted only to 3 or 4 methacrylates each. According to the authors, the reactions to THFMA were probably because of cross-allergy to other methacrylates.

 

Clinical records of patients with known occupational allergic contact dermatitis from methacrylates used in dentistry were analysed: Positive reactions to THFMA in the patch test were detected in 6/32 patients with wide methacrylate allergy. According to the authors, the reactions to THFMA were probably because of cross-allergy to other methacrylates.

 

There are no data gaps for the endpoint skin sensitisation.

 

 

Reference:

Bauch C, Kolle SN, Ramirez T, Eltze T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R., 2012. Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regul Toxicol Pharmacol. 63:489-504.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfilled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account.

Justification for classification or non-classification

Based on the available reliable, relevant and adequate data, THFMA is classified as skin sensitiser (Category 1, H317: May cause an allergic skin reaction) according to regulation (EC) 1272/2008.