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Description of key information

A 1974 patch study by Epstein on twenty-two healthy male inmate volunteers produced no reactions that were considered allergic.

In 2016 both In vitro Direct peptide reactivity assay's (DPRA) and In vitro KeratinoSens assay's were run on the test material. The In vitro DPRA assay resulted in a negative result and the In vitro KeratinoSens assay gave a positive result.

Subsequently, in 2017 an LLNA study was conducted. The SI values calculated for the test item concentrations 25, 50 and 100% were 2.1, 4.6 and 5.5, respectively. These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 34% was calculated.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: 4 = Not assignable Documentation insufficient for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
other: KLIGMAN, A. (1966) The Identification of Contact Allergens by Human Assay, III. The Maximisation Test: A Procedure for Screening and Rating Contact Sensitizers. THE JOURNAL OF INVESTIGATIVE DERMATOLOGY. 47 (5). p393-409.
Deviations:
not specified
GLP compliance:
no
Type of study:
patch test
Justification for non-LLNA method:
LLNA not available at time of testing.
Species:
human
Strain:
other: Homo sapiens
Sex:
male
Route:
epicutaneous, occlusive
Vehicle:
no data
Concentration / amount:
100%
Route:
epicutaneous, occlusive
Vehicle:
no data
Concentration / amount:
100%
No. of animals per dose:
22
Details on study design:
see below
Challenge controls:
Petrolatum (CAS 8009-03-8)
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
4
No. with + reactions:
0
Total no. in group:
22
Remarks on result:
other: dose is percentage
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
4
No. with + reactions:
0
Total no. in group:
22
Remarks on result:
other: dose is percentage

The data sheets with final tabulations are shown below. As can be seen from the data sheets, 1% SLS produced very little irritation. No evidence of contact sensitization was observed in the 22 test subjects.

Dimethyl Heptenal

Subject No. Challenge
SLS NO SLS SLS NO SLS
48-hrs 72-hrs 48-hrs 72-hrs 96-hrs 96-hrs
1 Caucasian 0 0 0 0    
2 Black 0 ± 0 0    
3 Caucasian 0 0 0 0    
4 Caucasian 0 0 0 0    
5 Black 0 0 0 0    
6 Black (Dropped - Now Show)
7 Black 0 0 0 0    
8 Caucasian (Paroled)
9 Caucasian 0 0 0 0    
10 Black 0 0 0 0    
11 Caucasian 0 ± 0 0    
12 Caucasian 0 B 0 0 B 0
13 Caucasian 0 B 0 0 B-H 0
14 Caucasian B 0 0 0    
15 Caucasian 0 0 0 0    
16 Caucasian (Dropped - W-Wing)
17 Caucasian 0 0 0 0    
18 Caucasian 1-B 0 0 0    
19 Caucasian 0 0 0 0    
20 Caucasian 0 0 0 0    
21 Caucasian B 0 0 0    
22 Caucasian 0 B 0 0 B 0
23 Caucasian 0 0 0 0    
24 Caucasian 0 ± 0 0    
25 Caucasian 0 0 0 0    

Petrolatum Control

Subject No. Challenge
SLS NO SLS SLS NO SLS
48-hrs 72-hrs 48-hrs 72-hrs 96-hrs 96-hrs
1 Caucasian     0 0    
2 Black     0 0    
3 Caucasian     0 0    
4 Caucasian     0 0    
5 Black     0 0    
6 Black (Dropped - Now Show)
7 Black     0 0    
8 Caucasian (Paroled)
9 Caucasian     0 0    
10 Black     0 0    
11 Caucasian     0 0    
12 Caucasian     0 0   0
13 Caucasian     0 0   0
14 Caucasian     0 0    
15 Caucasian     0 0    
16 Caucasian (Dropped - W-Wing)
17 Caucasian     0 0    
18 Caucasian     0 0    
19 Caucasian     0 0    
20 Caucasian     0 0    
21 Caucasian     0 0    
22 Caucasian     0 0   0
23 Caucasian     0 0    
24 Caucasian     0 0    
25 Caucasian     0 0    
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: not specified
Conclusions:
the test substance produced no reactions that were considered allergenic in the 22 subjects tested.
Executive summary:

A study was carried out on 22 healthy male inmate volunteers according to the method of Kligman (1966). The test substance was applied with and without pretreatment with SLS, and volunteers monitored for 72 hours, with some also being observed at 96 hours also. The results indicate no contact sensitzation was observed in the study.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 20 December 2016 and 24 January 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identification: Melonal
Appearance: Colourless to pale yellow liquid
Batch: PE0016691
Details on the study design:
N/A
Details on the study design:
N/A
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Mouse, CBA/J strain, inbred, SPF-Quality.
Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).
Source: Janvier, Le Genest-Saint-Isle, France
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 or 100%
No. of animals per dose:
5
Details on study design:
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test.
One group of five animals was treated with the vehicle.
Parameter:
SI
Value:
2.1
Test group / Remarks:
25% Concentration
Parameter:
SI
Value:
4.6
Test group / Remarks:
50% Concentration
Parameter:
SI
Value:
5.5
Test group / Remarks:
100% Concentration
Key result
Parameter:
EC3
Value:
34

Pre-screen Test

Very slight erythema was noted for the animals treated at 50% and 100% between Day 1 and Day 6.

Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

Based on these results, the highest test item concentration selected for the main study was a 100% concentration.

Main Study

Skin Reactions / Irritation

The very slight erythema of the ears as shown by several animals treated at 50% and all animals treated at 100% on Days 2 and/or 3 was considered not to have a toxicologically significant effect on the activity of the nodes.

Systemic Toxicity

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopic Examination of the Auricular Lymph Nodes and Surrounding Area

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values

Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 990, 2144 and 2591 DPM, respectively. The mean DPM/animal value for the vehicle control group was 467 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 2.1, 4.6 and 5.5, respectively.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The SI values calculated for the test item concentrations 25, 50 and 100% were 2.1, 4.6 and 5.5, respectively.
These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 34% was calculated.
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity (see Appendix 2 of study report).
Executive summary:

The SI values calculated for the test item concentrations 25, 50 and 100% were 2.1, 4.6 and 5.5, respectively.

These results indicate that the test item could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 34% was calculated.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May 17, 2016 to May 22, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
Principles of method if other than guideline:
The Direct Peptide Reactivity Assay (DPRA) is an in chemico test to determine the reactivity of test a substance towards peptides.
This assay has been validated for a broad range of low-molecular weight chemicals and it was found to detect reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA).
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
The test substance MELONAL was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by MELONAL was determined by HPLC-UV.
Positive control results:
As indicated in Table 7, all the acceptance criteria were fulfilled for the positive control cinnamic aldehyde.
Parameter:
other: Cys-peptide depletion
Value:
5.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Value is a percentage
Parameter:
other: Lys-peptide depletion
Value:
1.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Value is a percentage
Other effects / acceptance of results:
The test substance gave 5.1 % depletion of the Cys-peptide and 1.2 % depletion of the Lys-peptide. The average peptide depletion is 3.2 %. This is below the threshold of 6.38%, and the substance is thus attributed to the “minimal” reactivity class, rating it as a non-sensitizer according to the DPRA prediction model.
Acceptance criteria: The standard deviation for Cys-peptide depletion should be < 14.9% and for Lys-peptide depletion < 11.6%. These criteria were fulfilled (3.2% and 0.1% SD, respectively).
The co-elution controls indicated no co-elution with an UV-absorbing component.
   Average  Standard deviation
 Cys-peptide depletion  5.1  3.2
 Lys-peptide depletion  1.2  0.1
 Average depletion Cys-and Lys-peptide  3.2  
 Reactivity Class  MINIMAL  
 Prediction  Non-sensitizer  
 Elution time Cys peptide  10.64  
 Elution time test substance Cys peptide run  15.5  
 Elution time Lys peptide  7.97  
 Elution time test substance Lys peptide run  15.3  
Interpretation of results:
GHS criteria not met
Conclusions:
The result of the DPRA assay should be used as part of an integrated approach for testing and assessment (IATA). A parallel test in the KeratinoSens™ assay may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of chemicals, two congruent results in these two tests give a good prediction of the sensitizer hazard [3-5] particularly when predicting human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.
MELONAL was non-reactive and classified into the MINIMAL reactivity class according to the prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.
Executive summary:

MELONAL was non-reactive and classified into the minimal reactivity class according to the DPRA prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 14, 2016 to June 24, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
Principles of method if other than guideline:
The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response.
This assay has been validated for a broad range of low-molecular weight chemicals and it was found to respond to skin sensitizers from a broad range of so called applicability domains, ie. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approach for testing and assessment (IATA)
GLP compliance:
yes
Type of study:
activation of keratinocytes
Details on the study design:
The test substance MELONAL was dissolved in DMSO and tested according to the standard operating procedure of the KeratinoSensTM assay at 12 concentrations in three repetitions, each time in three replicates. After 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.
Positive control results:
Cinnamic aldehyde was run in all three repetitions. Here the detailed results for this positive control are reported in Table 8 and Figure 5. Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This was the case in all three repetitions. The induction at 64 µM and the EC 1.5 for cinnamic aldehyde were also calculated. The targets are: (i) Average induction in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8, and (ii) the EC 1.5 value should be between 7 µM and 30 µM. At least one of these two numerical criteria must be met in order to accept a repetition. In the experiments performed here both criteria were fulfilled in all three repetitions. Thus all three repetitions were valid for the positive control.
Run / experiment:
other: Rep 1
Parameter:
other: Cytotoxicity
Value:
880.15
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Result Value in μM
Run / experiment:
other: Rep 2
Parameter:
other: Cytotoxicity
Value:
774.56
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Result Value in μM
Run / experiment:
other: Rep 3
Parameter:
other: Cytotoxicity
Value:
684.83
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Result Value in μM
Run / experiment:
other: Rep 1
Parameter:
other: Luciferase
Value:
6.88
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Result Value is 'Imax fold-induction'
Run / experiment:
other: Rep 2
Parameter:
other: Luciferase
Value:
2.36
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Result Value is 'Imax fold-induction'
Run / experiment:
other: Rep 3
Parameter:
other: Luciferase
Value:
5.14
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Result Value is 'Imax fold-induction'

Cytotoxicity determinations.Given is the IC50 value as the concentration inµM reducing the viability by 50%.

 

Test substance

Rep 1 IC50 (µM)(*)

Rep 2 IC50 (µM)

Rep 3 IC50 (µM)

Geometric Mean

IC 50 (µM)

Standard deviation

IC 50 (µM)

1

MELONAL

880.15

774.56

684.83

775.77

97.76 

Luciferase determinations.Given is the Imaxvalues indicating maximal fold-induction up to a concentration of 1000µM.

 

Test substance

Rep 1 IMAX(fold induction)

Rep 2 IMAX(fold induction)

Rep 3 IMAX(fold induction)

Average IMAX(fold induction)

Standard deviation IMAX(fold induction)

1

MELONAL

6.88

2.36

5.14

4.79

2.28

Overall rating of the test substance according to the prediction model and the number of positive repetitions.

 

 

Reps pos.

Overall rating

1

MELONAL

2 of 3

POSITIVE

Interpretation of results:
study cannot be used for classification
Conclusions:
In all three repetitions induction of the luciferase above the threshold of 1.5 was noted. In two repetitions, induction occurred at non-cytotoxic levels (Viability > 70%). Melonal is thus rated positive in the KeratinoSens™ assay.
The result of the KeratinoSens™ assay should be used as part of an integrated approach for testing and assessment (IATA)[9]. A parallel test in the DPRA may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of substances, two congruent results in these two tests give a good prediction of the sensitizer hazard [5, 7, 19], in particular when comparing against human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.
Executive summary:

In all three repetitions induction of the luciferase above the threshold of 1.5 was noted. In two repetitions, induction occurred at non-cytotoxic levels (Viability > 70%). Melonal is thus rated positive in the KeratinoSens™ assay.

The result of the KeratinoSens™ assay should be used as part of an integrated approach for testing and assessment (IATA)[9]. A parallel test in the DPRA may indicate whether congruent results are obtained by both test methods. According to a detailed analysis on large set of substances, two congruent results in these two tests give a good prediction of the sensitizer hazard [5, 7, 19], in particular when comparing against human data, while an additional test in a dendritic cell line assessing expression of surface markers may be needed in case of discordant results.

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

A 1974 contact sensitising study by Epstein was carried out on 22 healthy male inmate volunteers according to the method of Kligman (1966). The test substance was applied with and without pretreatment with SLS, and volunteers monitored for 72 hours, with some also being observed at 96 hours also. The results indicate no contact sensitisation was observed in the study. Therefore it was concluded that through repetitive epicutaneous exposure there was no serious human health allergen activity.


Justification for selection of skin sensitisation endpoint:
Subsequently, a recent 2016 DPRA assay showed Melonal was non-reactive and classified into the minimal reactivity class according to the DPRA prediction model. It is therefore considered a non-sensitizer according to the prediction model of the DPRA.

However in a 2016 study Melonal was shown to be weakly toxic to the KeratinoSens™ cells. In two repetitions, it did induce the luciferase gene above a threshold of 1.5 at non-toxic concentrations. It is therefore considered a sensitizer according to the prediction model of the KeratinoSens™ assay.

As a result of these conflicting results a Local Lymph Node Asaay (LLNA) was performed on the test substance, the results from which indicated that the test item could elicit a SI≥ 3. Hence Melonal is considered as a sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

In an older human study, no sensitisation was observed. Subsequent more recent DPRA and KeratinoSens assays resulted in conflicting negative and a positive results respectively. Hence a LLNA study was performed. This resulted in a positive result with an SI value of 5.5 for the neat substance, hence the substance is classified as a sensitiser.