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Administrative data

Description of key information

human maximisation test, occlusive, induction (5 x 48 h), challenge (48 h) 10 d after induction, pre-treatment with 5 % SDS (induction and challenge): negative

DPRA, OECD 442C, GLP, mean peptide depletion: 24.85 % - positive (moderate reactivity)

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
human maximisation test: Substances are classified according to their allergenic capabilities after five 48 hour exposures of high concentrations to inflamed skin.
GLP compliance:
no
Type of study:
other: human maximisation test
Justification for non-LLNA method:
use of available data: study from 1972
Specific details on test material used for the study:
no details given
Species:
other: human
Sex:
male
Details on test animals and environmental conditions:
Twenty-five healthy male inmate volunteers. The volunteers were prisoners.
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Day(s)/duration:
48 h
Adequacy of induction:
other: non-irritant substance, but skin pre-treated with 10% SDS
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Day(s)/duration:
48 h
Adequacy of challenge:
other: challenge applications were preceded by one-hour applications of 10 % SDS under occlusion
No. of animals per dose:
25
Details on study design:
RANGE FINDING TESTS: The test item was pre-tested on five subjects in order to determine whether SDS pre-treatment was required. A patch of hexanal was applied to normal sites on the backs for 48 h under occlusion. No subject had any irritation and it was decided to use SDS pre-treatment in the test. Thus, the patch sites for the main test were pre-treated with SDS for 24 h with 5 % SDS.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 5
- Exposure period: 5 d
- Test groups: 25 volunteers
- Site: volar forearm
- Frequency of applications: 5 alternate days
- Duration: 48 h
- Concentrations: 100 %

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: challenge 10 days after induction
- Exposure period: 48 h
- Test groups: 25 volunteers
- Site: fresh sites (different from induction sites) pre-treated with 4 % SDS for 24 h
- Concentrations: 100 %
- Evaluation (hr after challenge): on removal and 24 h thereafter
Challenge controls:
not specified
Positive control substance(s):
not specified
Positive control results:
not specified
Key result
Reading:
1st reading
Hours after challenge:
0
Group:
test group
No. with + reactions:
0
Total no. in group:
25
Clinical observations:
not specified
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
No. with + reactions:
0
Total no. in group:
25
Clinical observations:
not specified
Remarks on result:
no indication of skin sensitisation
Group:
positive control
Remarks on result:
not measured/tested
Group:
negative control
Remarks on result:
not measured/tested
Interpretation of results:
GHS criteria not met
Conclusions:
There were no instances of contact-sensitization of hexanal in human maximisation test.
Executive summary:

Human maximisation test was performed on 25 volunteer prisoners. Induction was performed on 5 alternated days for 48 h (occlusive) on SDS pre-treated skin. Challenge was performed 10 days after induction for 48 h (occlusive) on SDS pre-treated skin. Reading was performed directly after challenge and 24 h thereafter. No volunteer was sensitized to hexanal in this test.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018/09/24 - 2018/10/26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
In experiment 3, the second set of reference controls B for Cys-peptide was not loaded in the HPLC at the time of programmed measurement and therefore the three replicates were measured 11 hours and 34 min after the end of analysis sequence. This can be seen as uncritical, because the reference control sets B1 and B2 were used as stability control for the peptide over the total analysis time. Even though they were measured later than necessary, values of set B2 were stable and met the acceptance criteria.
Deviations:
yes
Remarks:
please refer to remarks
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, 55116 Mainz
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
Based on the non-GLP pre-test, 100 mM test item solution was prepared by dissolving 30.2 mg (± 10 %) test item in 3 mL of the solvent acetonitrile.
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
A direct peptide reactivity assay (DPRA test) according to OECD 442C and GLP was performed with the test item. Three experiments were performed. The study was performed in order to evaluate the reactivity of the test item Hexanal towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile was incubated 22 h and 5 min for the Cys-peptide and 22 h for the Lys-peptide at 25 °C, respectively, and the peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

Peptides with ≥95 % purity, synthesized by Genecust, Dudelange, Luxemburg, were used.
Cys-Peptide (Cysteine)
Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Batch no.: P151223-TL472063
Purity: 98.12 %

Lys-Peptide (Lysine)
Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol)
Batch no.: P170415-2-LR569640
Purity: 98.85 %

Positive controls were treated identically as the test item. The following positive controls were used:
• Cinnamaldehyde (CAS 104-55-2, food grade ≥95 %, batch no. MKBT8955V) was used as 100 mM (± 10 %) solution in acetonitrile for the Cys-peptide.
• 2,3-Butanedione (CAS 431-03-8, ≥99 %, batch no. BCBS3560V) was used as 100 mM (± 10 %) solution in acetonitrile for the Lys-peptide
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments per-formed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing mid-range depletion for the lysine peptide.

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptides over the total analysis time. Set C was incubated and analysed together with the samples and was used for calculation of the peptide depletion.

Co-elution control
Sample prepared from the respective peptide buffer and the test item, but without peptide.
Positive control results:
Cys-depletion [%]:
Rep. 1) 75.44
Rep. 2) 75.77
Rep 3) 77.21
Mean: 76.14
SD: 0.94

Lys depletion [%]
1) 38.14
2) 37.76
3) 44.86
Mean: 40.25
SD: 3.99
Parameter:
other: Cys-depletion [%]
Run / experiment:
Replicate 1
Value:
22.2
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Cys-depletion [%]
Run / experiment:
Replicate 2
Value:
24.03
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Cys-depletion [%]
Run / experiment:
Replicate 3
Value:
26.06
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Cys-depletion [%]
Run / experiment:
mean
Value:
24.1
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Lys-depletion [%]
Run / experiment:
Replicate 1
Value:
23.76
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Lys-depletion [%]
Run / experiment:
Replicate 2
Value:
24.04
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Lys-depletion [%]
Run / experiment:
Replicate 3
Value:
29.01
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Lys-depletion [%]
Run / experiment:
mean
Value:
25.6
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: Cys- and Lys-depletion [%]
Run / experiment:
mean
Value:
24.85
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:
DEMONSTRATION OF TECHNICAL PROFICIENCY: The ten proficiency chemicals listed in the guideline were tested. All ten proficiency chemicals showed the expected DPRA prediction and eight out of the ten chemicals showed depletion values consistent with the classification reported in the OECD guideline (LAUS in-house study).

ACCEPTANCE OF RESULTS:

Calibration curve
Acceptance criterion: The r² of linear calibration should be > 0.99.
Assessment: The calibration curve was linear with acceptable coefficient of determination 0.99997 for the Cys-peptide and 0.99999 and for the Lys-peptide, respectively.

Solvent control
Acceptance criterion:
a) The mean peptide concentration of solvent control samples of sets A and C should be 0.50 ± 0.05 mM
b) The variation coefficient (relative standard deviation, RSD) of measured values of the nine samples from sets B1, B2 and C should be < 15 %
Assessment:
a) The mean peptide concentration of all solvent controls (Reference A and Reference C) were in the acceptable range of 0.50 ± 0.05 mM for both peptides
b) The variation coefficients (RSD) of the measured values of Reference controls B and C in acetonitrile were in the acceptable range with 0.6 % for the Cys-peptide and 0.5 % and for the Lys-Peptide, respectively.

Peptide depletion
Acceptance criteria
a) The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
b) The mean peptide depletion value for the positive control 2,3-butanedione should be 10 % - 45 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
c) The standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion
Assessment:
a) The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and < 14.9 %, respectively, for the Cys-peptide.
b) The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and < 11.6 %, respectively, for the Lys-peptide.
c) The standard deviation for the test item replicates was < 14.9 % for the percent cysteine depletion for the test item.
The standard deviation for the test item replicates was < 11.6 % for the percent lysine depletion for the test item.

Evaluation of results according to the Cysteine 1:10/Lysine 1:50 prediction model

Mean peptide depletion   [%]  Reactivity Class  DPRA Prediction
 0 - ≤ 6.38 Minimal  Negative
 > 6.38 - ≤ 22.62 Low  Positive
 > 22.62 - ≤42.47 Moderate  Positive
 > 42.47 - ≤ 100 High  Positive

The mean peptide depletion in the Cys-peptide and Lys-peptide assay was 24.85 %, therefore the test item was classified with:

DPRA Prediction: Positive

Reactivity class: Moderate

Conclusions:
All acceptance criteria were fulfilled; therefore, the test was considered valid. The DPRA prediction for the test item Hexanal was positive with reactivity class moderate according to the Cysteine 1:10/Lysine 1:50 prediction model. No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.
Executive summary:

A direct peptide reactivity assay (DPRA test) according to OECD 442C and GLP was performed with the test item. Three experiments were performed. The study was performed in order to evaluate the reactivity of the test item Hexanal towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile was incubated 22 h and 5 min for the Cys-peptide and 22 h for the Lys-peptide at 25 °C, respectively, and the peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

Experiment 1 was not valid, because the reference control A of the Cys-peptide was not in range and the relative standard deviation (RSD) of the controls B and C was too high. For the Lys-peptide the peptide-depletion of the positive control was out of range.

In Experiment 2 the reference control A and the peptide depletion of the positive control for the Cys-peptide was not in range and the relative standard deviation (RSD) of the controls B and C was too high. For the Lys-peptide experiment 2 was valid and the results are re-ported here.

The third experiment was valid for the Cys-peptide and the results are reported here.

The invalid experiments are not reported in this report, but the raw data are kept in the test facility in the GLP- archive.

As a conclusion, the DPRA prediction is “positive” with moderate reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item Hexanal possesses moderate skin sensitisation potential.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Human maximisation test was performed on 25 volunteer prisoners. Induction was performed on 5 alternated days for 48 h (occlusive) on SDS pre-treated skin. Challenge was performed 10 days after induction for 48 h (occlusive) on SDS pre-treated skin. Reading was performed directly after challenge and 24 h thereafter. None of the volunteers was sensitized to hexanal in this test.

A direct peptide reactivity assay (DPRA test) according to OECD 442C and GLP was performed with the test item. Three experiments were performed. The study was performed in order to evaluate the reactivity of the test item Hexanal towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile was incubated 22 h and 5 min for the Cys-peptide and 22 h for the Lys-peptide at 25 °C, respectively, and the peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

Experiment 1 was not valid, because the reference control A of the Cys-peptide was not in range and the relative standard deviation (RSD) of the controls B and C was too high. For the Lys-peptide the peptide-depletion of the positive control was out of range.

In Experiment 2 the reference control A and the peptide depletion of the positive control for the Cys-peptide was not in range and the relative standard deviation (RSD) of the controls B and C was too high. For the Lys-peptide experiment 2 was valid and the results are re-ported here.

The third experiment was valid for the Cys-peptide and the results are reported here.

The invalid experiments are not reported in this report, but the raw data are kept in the test facility in the GLP- archive.

As a conclusion, the DPRA prediction is “positive” with moderate reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item Hexanal possesses moderate skin sensitisation potential.

Justification for classification or non-classification

A positive results was obtained in the OECD 442c study demonstrating, that the substance is able to bind to protein moietys. This is not really surprising for an aldehyde. However, the available skin sensitisation tests in human volunteers applying irritating concentrations for the induction clearly shows that Hexanal tested at 1 % (concentration limited due to irritating effects in combination with SDS)  does not cause skin sensitising effects in humans. Further in vitro tests are therefore of no value.

Based on the available information, it is concluded that the substance does not need to be classified according to Regulation (EC) No 1272/2008.