Registration Dossier

Administrative data

Description of key information

3 -methylbutan-1 -ol which did not cause sensitization in humans. Furthermore, it is not peptide reactive and does not activate keratinocytes.

This matches the results for Pentan-1 -ol. Pentan-1 -ol is not peptide reactive and does not activate keratinocytes.

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:
weight of evidence
Study period:
08-11-2017 - 11-20-2017
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))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
The analyses of the test item (= test substance) were carried out at Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany.
Name of test substance: 3-Methyl-1-butanol
Test-substance No.: 11/0557-2
Batch identification: 59086375L0
CAS No.: 123-51-3
Purity: 99.7 corrected area-% (final report study code: 17L00189). For the DPRA 99.8% was used for calculation of 100 mM concentration, as this value was reported as preliminary information to study code 17L00189.
Identity: confirmed (final report study code: 17L00189)
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this
responsibility. The test facility is organizationally independent
from the BASF SE sponsor division.

ADDITIONAL TEST-SUBSTANCE INFORMATION
Physical state / color: liquid / colorless, clear
Storage conditions: ambient
Molecular weight: 88.15 g/mol
Log KOW: 1.28 (information taken from safety data sheet)

Proposed reaction mechanism for protein binding by OECD toolbox (non-GLP system):
The OECD toolbox did not indicate an alert for protein binding for either the substance or its predicted metabolites (auto-oxidation, hydrolysis, and skin metabolism).

Because the test-substance preparation was incubated with the peptide (DPRA), no analysis of the test substance in the vehicle was performed.
Details on study design:
TEST SYSTEM
Synthetic peptides: Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and JPT Peptide Technologies GmbH, Berlin, Germany)
containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.


Controls for the DPRA
Negative control (NC): vehicle control = acetonitrile
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as a 50 mM solution in acetonitrile.
Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Test substance preparation for the DPRA
The test substance was prepared as a 100 mM (considering a molecular weight of 88.15 g/mol and a purity/contents of 99.8% (preliminary information to study code 17L00189)3) preparation in acetonitrile. After short stirring the test substance was soluble in the vehicle.
Vehicle: acetonitrile
Reason for the vehicle: The test substance was soluble in acetonitrile.

Experimental procedure of the DPRA
The test substance was dissolved in a suitable vehicle. Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (=NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.
Test substance solubility
Prior to the assay the solubility of the test substance at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test-substance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

Preparation of peptide stock solutions
Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test-substance and control samples.
Preparation of calibration samples
The following calibration samples were prepared from the peptide stock solutions in 20% acetonitrile in the respective buffer (= dilution buffer) using serial dilution:
Calib. 1 Calib. 2 Calib. 3 Calib. 4 Calib. 5 Calib. 6 Dilution buffer
mM peptide 0.534 0.267 0.134 0.067 0.033 0.017 0.000
The analysis of the calibration samples was started before analysis of the test-substance samples.

Preparation of the test-substance samples
The samples were prepared in triplicates per concentration for each peptide according to the pipetting scheme given below.
C-peptide: 750 μL C-peptide stock-solution; 200 μL solvent (vehicle); 50 μL test-substance preparation (or PC-preparation or solvent (VC))
K-peptide: 750 μL K-peptide stock-solution; 250 μL test-substance preparation (or PC-preparation or solvent (VC))

The samples were prepared in suitable tubes and capped tightly and were incubated in the dark for 24 ± 2 hours. They should be incubated at 25°C ± 2.5°C. Due to a technical error of the incubator the actual incubation temperature was 29.6°C. Hence, the incubation temperature given in the OECD TG 442C was exceeded by ca. 2°C. However, this deviation does not influence the result of the DPRA as was demonstrated in an additional study (see project 64V0478/99V001). Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

Preparation of the vehicle controls
Several vehicle controls were prepared in triplicates in the same way as the test-substance samples described above but with the vehicle (acetonitrile) instead of the test substance: One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

Preparation of the co-elution control
One sample per peptide was prepared in the same way as the test-substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

Measurement of peptide concentrations
The analyses of the samples were performed via HPLC under the following conditions:
Column: ZORBAX SB-C18 2.1 x 100 mm, 3.5 μm with guard column SecurityGuard Ultra Cartridges, UHPLC C18 for 4.6 mm ID (Phenomenex)
Mobile phase: A: H2O/ACN/TFA 950/50/1 V/V/V
B: ACN/H2O/TFA 950/50/0.85 V/V/V
Flow: 0.50 mL/min
Gradient:
time [min] %B
0 5
8 20
8.1 90
10 90
10. 1 5
16 5
Wavelength: 220 nm and 258 nm
Injection volume: 2 μL
Software: Dionex Chromeleon

Acceptance criteria of the DPRA
The standard calibration curve should have an r² >0.99.
The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
The CV of the nine vehicle controls B and C should be < 15%.
Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).
In addition the positive control should cause depletion of both peptides comparable to historic data.

Evaluation of results of the DPRA
Peptide depletion
Chemical reactivity was determined by mean peptide depletion [%] and was rated as high, moderate, low, or minimal:

Evaluation criteria of DPRA; cysteine 1:10 / lysine 1:50 prediction model:
Mean peptide depletion[%] Reactivity Evaluation
> 42.47 high reactivity positive
> 22.62 ≤ 42.47 moderate reactivity positive
> 6.38 ≤ 22.62 low reactivity positive
≤ 6.38 minimal or no reactivity negative

In the case mean peptide depletion [%] cannot be determined due to invalid K-peptide depletion (e.g. insolubility of the K-peptide samples or interference in the samples of the K-peptide) but valid C-peptide depletion is available, evaluation is performed as follows:

Evaluation criteria of DPRA; cysteine 1:10 prediction model:
C peptide depletion [%] Reactivity Evaluation
> 98.24 high reactivity positive
> 23.09 ≤ 98.24 moderate reactivity positive
> 13.89 ≤ 23.09 low reactivity positive
≤ 13.89 minimal or no reactivity negative

Limitations of the evaluation by insolubility and gravimetric procedure
For test substances that are not completely soluble by visual observation in the sample preparations containing the peptides immediately after preparation or after 24 hours, or when a gravimetric procedure is applied (with the exception of application of the undiluted test substance (liquids) or the maximal soluble test-substance concentration (solids)), the result may be under-predictive due to limited availablity of the test substance. In this case mean peptide reactivity ≤ 6.38% (cysteine 1:10 / lysine 1:50 prediction model) or ≤ 13.89% (cysteine 1:10 prediction model) is interpreted as “inconclusive”. However, a mean peptide depletion > 6.38% or > 13.89% is considered as “positive”.
Positive control results:
see section "Any other information on results incl. tables"
Key result
Parameter:
other: Cystein-Peptide depletion [%]
Value:
0.48
Vehicle controls validity:
valid
Remarks:
comparable to historic data
Negative controls validity:
valid
Remarks:
Vehicle (ACN) was used as negative control
Positive controls validity:
valid
Remarks:
comparable to historic data
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Lysine-Peptide depletion [%]
Value:
-0.14
Vehicle controls validity:
valid
Remarks:
comparable to historic data
Negative controls validity:
valid
Remarks:
Vehicle (ACN) was used as negative control
Positive controls validity:
valid
Remarks:
comparable to historic data
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Solubility of the test-substance samples with the peptides
The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.

Co-elution
No co-elution of the test substance and peptides occurred as demonstrated by the consistent values of the area ratios 220 nm/258 nm and chromatograms of the co-elution control.

Cysteine-peptide vehicle controls in acetonitrile

The mean peptide concentration of the three samples of set A was calculated to be 0.502 mM with a SD of 0.005 mM, demonstrating good performance.

The mean peptide concentration of the three samples of set B, analyzed at the beginning of thesamplelistwascalculatedtobe0.467mMwithaSDof0.043mM.Theotherthreesamples of set B, analyzed at the end of the sample list had a mean peptide concentration of 0.477 mM with a SD of 0.002 mM.

The CV of the 9 vehicle control samples of sets B and C was calculated to be 5.0%. Thus the peptide was considered stable over the time of analysis. Reaction withcysteine-peptide

 

DPRA Peak area, peptide concentration and peptide depletion of NC, PC and the test substance for cysteine-peptide.

 

 

Reaction with cysteine- peptide

peak area [mAU*s]

at 220 nm

 

peptide concentration [mM]

sample 1

sample 2

sample 3

sample 1

sample 2

sample 3

mean          SD

 

NC: ACN

 

486.2

 

483.0

 

474.0

 

0.494

 

0.491

 

0.482

 

0.489       0.006

 

3-Methyl-1-butanol

 

484.3

 

478.8

 

473.1

 

0.492

 

0.486

 

0.481

 

0.486       0.006

 

PC: EGDMA in ACN

 

198.5

 

186.5

 

183.0

 

0.202

 

0.190

 

0.187

 

0.193       0.008

 

 

Reaction with cysteine- peptide

 

peptide depletion [%]

sample 1

sample 2

sample 3

mean     sD

 

NC: ACN

 

-1.07

 

-0.40

 

1.47

 

0.00         1.31

 

3-Methyl-1-butanol

 

-0.67

 

0.46

 

1.65

 

0.48        1.16

 

PC: EGDMA in ACN

 

58.57

 

61.05

 

61.77

 

60.46     1.68

 

 

 

 

DPRA. Area ratio 220 nm/258 nm of NC, PC and the test substance for cysteine-peptide.

 

 

Reaction with cysteine- peptide

peak area [mAU*s]

at 258 nm

 

area ratio 220 nm/258 nm

sample 1

sample 2

sample 3

sample 1

sample 2

sample 3

 

NC: ACN

 

16.1

 

15.8

 

15.6

 

30.3

 

30.6

 

30.3

 

3-Methyl-1-butanol

 

15.9

 

15.8

 

15.4

 

30.4

 

30.3

 

30.7

 

PC: EGDMA in ACN

 

6.5

 

6.1

 

6.0

 

30.6

 

30.5

 

30.5

 

The mean area ratio 220 nm/ 258 nm of the 9 vehicle control samples of sets B and C was calculated to be 30.7. Hence, the area ratio 220 nm/ 258 nm of the test substance samples correspond to 98.6% to 100.1% of the mean of the vehicle controls.

 Lysine-peptide vehicle controls inacetonitrile

The mean peptide concentration of the three samples of set A was calculated to be 0.499 mM with a SD of 0.001 mM, demonstrating good performance.

The mean peptide concentration of the three samples of set B, analyzed at the beginning of thesamplelistwascalculatedtobe0.498mMwithaSDof0.000mM.Theotherthreesamples of set B, analyzed at the end of the sample list had a mean peptide concentration of 0.497 mM with a SD of 0.001 mM. The CV of the 9 vehicle control samples of sets B and C was calculated to be 0.1%. Thus the peptide was considered stable over the time of analysis.

Reaction withlysine-peptide

 

DPRA. Peakarea, peptide concentration and peptide depletion of NC, PC and the test substance for lysine-peptide.

 

 

Reaction with lysine- peptide

peak area [mAU*s]

at 220 nm

 

peptide concentration [mM]

sample 1

sample 2

sample 3

sample 1

sample 2

sample 3

mean          SD

 

NC: ACN

 

479.7

 

478.5

 

477.6

 

0.499

 

0.497

 

0.496

 

0.497       0.001

 

3-Methyl-1-butanol

 

481.0

 

479.2

 

477.7

 

0.500

 

0.498

 

0.496

 

0.498       0.002

 

PC: EGDMA in ACN

 

431.0

 

431.5

 

443.5

 

0.448

 

0.448

 

0.461

 

0.452       0.007

 

 

Reaction with lysine- peptide

 

peptide depletion [%]

sample 1

sample 2

sample 3

mean       SD

 

NC: ACN

 

-0.23

 

0.03

 

0.21

 

0.00         0.22

 

3-Methyl-1-butanol

 

-0.49

 

-0.12

 

0.19

 

-0.14        0.34

 

PC: EGDMA in ACN

 

9.97

 

9.88

 

7.35

 

9.07         1.49

 

 

 

DPRA. Area ratio 220 nm/258 nm of NC, PC and the test substance for lysine-peptide.

 

 

Reaction with lysine- peptide

peak area [mAU*s]

at 258 nm

 

area ratio 220 nm/258 nm

sample 1

sample 2

sample 3

sample 1

sample 2

sample 3

 

NC: ACN

 

14.8

 

14.9

 

14.7

 

32.3

 

32.0

 

32.4

 

3-Methyl-1-butanol

 

15.2

 

14.9

 

14.6

 

31.7

 

32.1

 

32.8

 

PC: EGDMA in ACN

 

13.4

 

13.2

 

13.7

 

32.2

 

32.6

 

32.3

 

 

The mean area ratio 220 nm/ 258 nm of the 9 vehicle control samples of sets B and C was calculated to be 32.3. Hence, the area ratio 220 nm/ 258 nm of the test substance samples correspond to 98.2% to 101.6% of the mean of the vehicle controls.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results and applying the evaluation criteria described in chapter 3.10, 3-Methyl-1-butanol is not peptide reactive and does not activate keratinocytes. Applying the evaluation criteria described in chapter 3.10 3-Methyl-1-butanol is predicted not to be a skin sensitizer.
Executive summary:

Testing 3-Methyl-1-butanol in two different non-animal methods addressing different key events of the skin sensitization Adverse Outcome Pathway resulted in two negative results in the DPRA and LuSens.

The three tests generally used in the “2 out of 3” ITS assess protein binding in chemico (DPRA), triggering an antioxidant response in keratinocytes (LuSens) and activation of dendritic cells (h-CLAT). Of note,the molecular initiating event of protein binding also occurs in the cell-based assays (LuSens, h-CLAT) and is not solely detected via the DPRA.

 PREDICTIVITY

The common outcome of the approach is a consistent prediction in all contributing assays. Cases, where one assay gives a different prediction than the other two are not in conflict with the Adverse Outcome Pathway but reflect known limitations of in-vitro/in-chemico systems. Indeed, judging a test substance as a sensitizer based on only one positive test results in poor predictivity (Urbisch et al.,2015). The predictivities of the single assays as well as the “2 out of 3” ITS was compared to both animal and human data (Table21, Urbisch et al., 2015). It achieved accuracies of 90% or 79% compared to human (n = 60 to 101) or LLNA (n =213) data, respectively. A more accurate prediction of the skin sensitization potential in humans is achieved than by performing the LLNA.

 

Sensitivity, specificity and predictive accuracy of single non-animal test methods and the “2 out of 3” ITS compared to human data (Urbisch et al. 2015).

 

Sensitivity [%]

Specificity [%]

Accuracy [%]

n

2 out of 3 ITS

90

90

90

101

DPRA

84

84

84

102

LuSens

78

79

79

60

h-CLAT

89

64

82

98

 

The OECD Toolbox/TIMES-SS did not indicate a specific protein-binding alert for3-Methyl-1- butanol. The predictive accuracy of the skin sensitization potential of substances without an alert for an obvious reaction mechanism was 80% (Urbisch et al. 2015).

 

APPLICABILITY

Chemical similarity of the test substance with the validation dataset

To provide an estimate for the chemical similarity of the test substance assessed in this study and substances with available in vitro and in vivo data (published in Urbisch et al., 2015), a nearest neighbor analysis has been conducted. 


 UNCERTAINTYANALYSIS

 Uncertainty arising from technical and biologicalvariance

The borderline range depicts the variance of the individual test methods, including technical and biological variability (Leontaridou et al., 2017). It addresses the uncertainty of the three assays around their respective classification thresholds and represents a range in which the likelihood to obtain a positive or negative result just below or above the classification threshold is equal. This range was determined for each assay statistically (pooled standard deviations), using historic BASF data (Leontaridou et al., 2017). It is useful especially for assays for which no individual statistical analysis is possible due to low number of replicates per treatment (e.g. h- CLAT and DPRA). This evaluation is an amendment to the evaluation given in respective OECD Test Guidelines.

The definition of a borderline range allows the possible prediction as “ambiguous”.

 

Pooled standard deviations and borderline ranges (Leontaridou et al., 2017).Test results in the range of 3.38%≤MPD≤9.38% (DPRA), FI≤3 (LuSens) and CD86FI≤3 as well as CD54FI≤3 (h-CLAT) were considered for quantifying the pooled standard deviation. FI = fold induction; MPD = mean peptide depletion.

Pooled standard deviation

Borderline range

DPRA, cysteine 1:10 / lysine 1:50

DPRA, cysteine 1:105

1.52%

3.39%

MPD = {4.86%, 7.90%}

MPD = {10.50%; 13.89%}

LuSens

0.229

FI = {1.27, 1.73}

h-CLAT CD86 h-CLAT CD54

26%

19%

CD86 increase = {124%, 176%}

CD54 increase = {181%, 219%}

 

In the present study, none of the performed single assay met the “borderline”-criteria and the results are therefore considered to be unambiguous.

 

METABOLIC CAPACITY

The test system is able to detect most pre- and pro-haptens and a negative prediction is considered acceptable. In vitro investigations (Urbisch et al., 2016 and Patlewicz et al., 2016) using compounds requiring molecular transformation to attain a sensitizing potential have shown that pre-haptens can readily be detected in the DPRA, many of which involve autoxidation processes. Moreover, many pro-haptens are also activated by non-enzymatic oxidation (and therefore are pre-andpro-haptens). The cellular models h-CLAT and LuSens have been shown to detect pro-haptens more efficiently; respective enzyme activities were detected in the cell lines (Fabian et al., 2013). Thus, within the scope of this study, potentially relevant molecular transformations have been considered.

Supporting information on the role of metabolites was obtained via the skin metabolism simulator of the OECD Toolbox, noting that this is an expert rule system which – in the general absence of experimental skin metabolism data – has not been validated against such data. For the present test substance, metabolites were identified and none of them have a structural alert for protein binding. The relevance of the obtained data with respect to the potential of the test systems to detect pre- and pro-haptens is considered as given.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
08-11-2017 - 11-20-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
The analyses of the test item (= test substance) were carried out at Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany.
Name of test substance: 3-Methyl-1-butanol
Test-substance No.: 11/0557-2
Batch identification: 59086375L0
CAS No.: 123-51-3
Purity: 99.7 corrected area-% (final report study code: 17L00189). 99.7% was used for calculation of final test-substance
concentrations in the LuSens.
Identity: confirmed (final report study code: 17L00189)
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this
responsibility. The test facility is organizationally independent
from the BASF SE sponsor division.

ADDITIONAL TEST-SUBSTANCE INFORMATION
Physical state / color: liquid / colorless, clear
Storage conditions: ambient
Molecular weight: 88.15 g/mol
Log KOW: 1.28 (information taken from safety data sheet)

Proposed reaction mechanism for protein binding by OECD toolbox (non-GLP system):
The OECD toolbox did not indicate an alert for protein binding for either the substance or its predicted metabolites (auto-oxidation, hydrolysis, and skin metabolism).
Because the test-substance preparation was applied shortly after preparation, no analysis of the test substance in the vehicle was
performed.
Details on study design:
Cell line: LuSens
Human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen, Germany.

Controls for the LuSens
Negative control (NC): DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 μM (= 450 μg/mL) in 1% DMSO in culture medium 3
Positive control (PC): ethylene glycol dimethacrylate (EGDMA, CAS no.: 97-90-5), 90.8 μM (= 18 μg/mL) in 1% DMSO in culture medium 3
Vehicle control (VC): 1% DMSO in culture medium 3
Blank control: Culture medium 3 without cells
Basal control: Culture medium 3 with cells

Test substance preparation for the LuSens
The test substance was weighed and topped up with the chosen vehicle (DMSO) to achieve the required 100x concentration of the highest concentration (stock solution). Further concentrations were prepared as 100x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate) and were further diluted (1:25) in culture medium 3 to obtain 4x concentrations (dilution plate). The test-substance preparations were prepared by stirring.
Reason for the vehicle: The test substance was soluble in DMSO.
Form of application: Visual inspection of each dilution step was performed.

Experimental procedure of the LuSens
Preparation of the cells
LuSens cells from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) for at least passage ≥5 but not longer than 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 105 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. Two independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.

Test-substance application for MTT and luciferase assay
After cell adaption for 24 hours cell culture medium 2 was aspirated and replaced with 150 μL medium 3. The test substance was prepared as described above. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%).

After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability.

Visual inspections
Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates.

Luciferase assay
After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Subsequently 200 μL of One-Glo-preparation (= 100 μL One-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

Cell viability assay MTT
Cell culture medium was aspirated from all wells. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and medium 3) was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

Acceptance criteria of the LuSens
A tested concentration is not further evaluated when relative viability is less than 70%.
The cell viability of VC cells must yield at least 85%.
The mean of the positive control EGDMA should achieve ≥2.50 fold-induction and the mean of the LA <1.50 and the mean of the viability must be ≥70%.
The CV [%] of the luminescence in the vehicle control wells for each plate should be below 20%.
The mean of the basal expression of the cells must be <1.50 fold-induction as compared to the solvent control.
In addition, positive, negative and vehicle control data should lie within the range of the historic data.

Evaluation of results of the LuSens
A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold-induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least two consecutive concentrations of two independent experiments. A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 2000 μM if molecular weight is applicable or 2000 μg/mL if molecular weight is not applicable) or maximum applicable concentration or up to the cytotoxicity limit (at least one concentration displaying viability below 70%). To be relevant for evaluation, the cell viability must be more than 70% in at least three tested concentrations of an experiment.

Limitations of the evaluation of the LuSens
As described in the publication of Urbisch et al., 2015 the result of the LuSens for acylating agents may be under-predictive. In the case a test substance is predicted to be an acylating agent, a “negative” result is interpreted as “inconclusive”. However, a “positive” result will be accepted.
Positive control results:
see section "Any other informtation on results incl. tables"
Key result
Parameter:
other: EC1.50 (the concentration resulting in a 1.50-fold luciferase induction)
Remarks:
was not applicable
Vehicle controls validity:
valid
Remarks:
comparable to historic data
Negative controls validity:
valid
Remarks:
comparable to historic data
Positive controls validity:
valid
Remarks:
comparable to historic data
Remarks on result:
no indication of skin sensitisation

LuSens. Mean values and standard deviations of luciferase induction and rel. viability as well as p- values of t-test (experiment 2). Concentrations with fold inductions above 1.50 with rel. viability ≥70% and with statistical significance are indicated in bold and in grey when < 70% viability.

 

Concentration

(test substance) [µM]

 

foldindu

 

ction

2ndexperiment

rel. viability [%]

 

 

t-test

mean

SD

mean

SD

p-value

markers

 

560

 

1.03

 

0.16

 

80

 

10

 

0.373

 

n.s.

672

1.08

0.13

72

10

0.191

n.s.

806

1.09

0.14

69

4

0.190

n.s.

967

1.01

0.12

76

8

0.474

n.s.

1161

1.01

0.07

79

13

0.396

n.s.

1393

1.09

0.06

79

10

0.049

*

1672

1.05

0.08

79

11

0.206

n.s.

2006

1.02

0.05

66

7

0.288

n.s.

VC

1.00

0.08

100

5

-

-

EGDMA 90.8 µM

5.19

0.36

83

8

0.000

**

LA 5000 µM

0.90

0.12

107

1

0.069

n.s.

LuSens. Mean values and standard deviations of luciferase induction and rel. viability as well as p- values of t-test (experiment 3). Concentrations with fold inductions above 1.50 with rel. viability ≥70% and with statistical significance are indicated in bold and in grey when < 70% viability.

 

Concentration(testsubstance)

[µM]

 

foldindu

 

ction

3rdexperiment

rel. viability [%]

 

 

t-test

mean

SD

mean

SD

p-value

markers

 

560

 

0.97

 

0.09

 

84

 

6

 

0.291

 

n.s.

672

0.95

0.07

84

4

0.181

n.s.

806

0.96

0.06

87

1

0.185

n.s.

967

0.93

0.04

88

2

0.038

*

1161

1.00

0.14

81

5

0.480

n.s.

1393

0.99

0.08

82

5

0.401

n.s.

1672

0.95

0.08

79

5

0.197

n.s.

2006

1.00

0.14

72

3

0.487

n.s.

VC

1.00

0.12

100

6

-

-

EGDMA 90.8 µM

5.33

0.47

80

8

0.000

**

LA 5000 µM

0.88

0.13

105

4

0.053

n.s.

The 1stexperiment is invalid for not meeting acceptance criteria and is not included in the report.

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results and applying the evaluation criteria, 3-Methyl-1-butanol does not activate keratinocytes. Applying the evaluation criteria-Methyl-1-butanol is predicted not to be a skin sensitizer.
Executive summary:

Testing 3-Methyl-1-butanol in two different non-animal methods addressing different key events of the skin sensitization Adverse Outcome Pathway resulted in two negative results in the DPRA and LuSens.

The three tests generally used in the “2 out of 3” ITS assess protein binding in chemico (DPRA), triggering an antioxidant response in keratinocytes (LuSens) and activation of dendritic cells (h-CLAT). Of note,the molecular initiating event of protein binding also occurs in the cell-based assays (LuSens, h-CLAT) and is not solely detected via the DPRA.

 PREDICTIVITY

The common outcome of the approach is a consistent prediction in all contributing assays. Cases, where one assay gives a different prediction than the other two are not in conflict with the Adverse Outcome Pathway but reflect known limitations of in-vitro/in-chemico systems. Indeed, judging a test substance as a sensitizer based on only one positive test results in poor predictivity (Urbisch et al.,2015). The predictivities of the single assays as well as the “2 out of 3” ITS was compared to both animal and human data (Table21, Urbisch et al., 2015). It achieved accuracies of 90% or 79% compared to human (n = 60 to 101) or LLNA (n =213) data, respectively. A more accurate prediction of the skin sensitization potential in humans is achieved than by performing the LLNA.

 

Sensitivity, specificity and predictive accuracy of single non-animal test methods and the “2 out of 3” ITS compared to human data (Urbisch et al. 2015).

 

Sensitivity [%]

Specificity [%]

Accuracy [%]

n

2 out of 3 ITS

90

90

90

101

DPRA

84

84

84

102

LuSens

78

79

79

60

h-CLAT

89

64

82

98

 

The OECD Toolbox/TIMES-SS did not indicate a specific protein-binding alert for 3-Methyl-1- butanol. The predictive accuracy of the skin sensitization potential of substances without an alert for an obvious reaction mechanism was 80% (Urbisch et al. 2015).

 

APPLICABILITY

Chemical similarity of the test substance with the validation dataset

To provide an estimate for the chemical similarity of the test substance assessed in this study and substances with available in vitro and in vivo data (published in Urbisch et al., 2015), a nearest neighbor analysis has been conducted. 


 UNCERTAINTYANALYSIS

 Uncertainty arising from technical and biologicalvariance

The borderline range depicts the variance of the individual test methods, including technical and biological variability (Leontaridou et al., 2017). It addresses the uncertainty of the three assays around their respective classification thresholds and represents a range in which the likelihood to obtain a positive or negative result just below or above the classification threshold is equal. This range was determined for each assay statistically (pooled standard deviations), using historic BASF data (Leontaridou et al., 2017). It is useful especially for assays for which no individual statistical analysis is possible due to low number of replicates per treatment (e.g. h- CLAT and DPRA). This evaluation is an amendment to the evaluation given in respective OECD Test Guidelines.

The definition of a borderline range allows the possible prediction as “ambiguous”.

 

Pooled standard deviations and borderline ranges (Leontaridou et al., 2017).Test results in the range of 3.38%≤MPD≤9.38% (DPRA), FI≤3 (LuSens) and CD86FI≤3 as well as CD54FI≤3 (h-CLAT) were considered for quantifying the pooled standard deviation. FI = fold induction; MPD = mean peptide depletion.

Pooled standard deviation

Borderline range

DPRA, cysteine 1:10 / lysine 1:50

DPRA, cysteine 1:105

1.52%

3.39%

MPD = {4.86%, 7.90%}

MPD = {10.50%; 13.89%}

LuSens

0.229

FI = {1.27, 1.73}

h-CLAT CD86 h-CLAT CD54

26%

19%

CD86 increase = {124%, 176%}

CD54 increase = {181%, 219%}

 

In the present study, none of the performed single assay met the “borderline”-criteria and the results are therefore considered to be unambiguous.

 

METABOLIC CAPACITY

The test system is able to detect most pre- and pro-haptens and a negative prediction is considered acceptable. In vitro investigations (Urbisch et al., 2016 and Patlewicz et al., 2016) using compounds requiring molecular transformation to attain a sensitizing potential have shown that pre-haptens can readily be detected in the DPRA, many of which involve autoxidation processes. Moreover, many pro-haptens are also activated by non-enzymatic oxidation (and therefore are pre-andpro-haptens). The cellular models h-CLAT and LuSens have been shown to detect pro-haptens more efficiently; respective enzyme activities were detected in the cell lines (Fabian et al., 2013). Thus, within the scope of this study, potentially relevant molecular transformations have been considered.

Supporting information on the role of metabolites was obtained via the skin metabolism simulator of the OECD Toolbox, noting that this is an expert rule system which – in the general absence of experimental skin metabolism data – has not been validated against such data. For the present test substance, metabolites were identified and none of them have a structural alert for protein binding. The relevance of the obtained data with respect to the potential of the test systems to detect pre- and pro-haptens is considered as given.

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

There is a In vitro Test Strategy Study available to test the skin sensitising potetial of 3 -Methyl-1 -butanol:

The objective was to assess the skin sensitizing potential of 3-Methyl-1-butanol. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

·        protein reactivity (DPRA),

·        activation of keratinocytes (LuSens), and

·        activation of dendritic cells (h-CLAT).

However, in the current case for 3-Methyl-1-butanol the results derived with DPRA and LuSens were sufficient for a final assessment. Therefore further testing in h-CLAT was waived. 

DPRA: The reactivity of 3 -Methyl-1 -butanol towards synthetic cysteine (C)- or lysine (K)- containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose,the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220nm.

The test substance was dissolved 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-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with thepeptides.

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. Moreover, the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220nm/258nm was calculated as a measure of peakpurity.

The following results were obtained in the DPRA:

The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.

No co-elution of test substance and peptides was present.

The mean C-peptide depletion, caused by the test substance was determined to be 0.48%. The mean K-peptide depletion, caused by the test substance was determined to be -0.14%.


Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.24%.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that 3 -Methyl-1 -butanol shows minimal chemical reactivity in the DPRA under the test conditions chosen. 

LuSens: The keratinocyte activating potential of test substance 3-Methyl-1-butanolwas evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. No cytotoxicity was observed. In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance.A total of 2 valid experiments were performed. The following results wereobserved:

The test substance was soluble in DMSO (100 x stock preparations) and in 1% DMSO in culturemedium3(final concentrations). No precipitates were noticed in any concentration after 48hours.

Relative luciferase fold induction and concurrent relative viability determined in the main experiments are summarized in table 1.

Table1: Summary of LuSens MainExperiments. 2 valid experiments (2nd and 3rd experiment) were performed. The 1stexperiment is invalid for not meeting acceptance criteria and is not included in the report. Concentrations with fold inductions above 1.50 with rel.viability≥70% and with statistical significance are indicated in bold and in grey when < 70%viability.

 

Concentration

(test substance) [µM]

 

fold induction

2ndexperiment

rel. viability [%

 

t-test

Concentration

(test substance) [µM]

 

fold induction

3rdexperiment

rel. viability [%

 

t-test

mean

mean

p-value

markers

mean

mean

p-value

markers

 

560

 

1.03

 

80

 

0.373

 

n.s.

 

560

 

0.97

 

84

 

0.291

 

n.s.

672

1.08

72

0.191

n.s.

672

0.95

84

0.181

n.s.

806

1.09

69

0.190

n.s.

806

0.96

87

0.185

n.s.

967

1.01

76

0.474

n.s.

967

0.93

88

0.038

*

1161

1.01

79

0.396

n.s.

1161

1.00

81

0.480

n.s.

1393

1.09

79

0.049

*

1393

0.99

82

0.401

n.s.

1672

1.05

79

0.206

n.s.

1672

0.95

79

0.197

n.s.

2006

1.02

66

0.288

n.s.

2006

1.00

72

0.487

n.s.

VC

1.00

100

-

-

VC

1.00

100

-

-

EGDMA 90.8 µM

5.19

83

0.000

**

EGDMA 90.8 µM

5.33

80

0.000

**

LA 5000 µM

0.90

107

0.069

n.s.

LA 5000 µM

0.88

105

0.053

n.s.

 Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable.

In summary, after 48 hours of exposure to test substance 3 -Methyl-1 -butanol luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance 3-Methyl-1-butanol does not have a keratinocyte activating potential.

 Table 2: Summary of individual test results.

 

Test Method

Test Result

Test Evaluation

Direct Peptide Reactivity Assay (DPRA)

0.24% mean peptide depletion (0.48%

cysteine peptide depletion; -0.14% lysine peptide depletion).a

Negative

Keratinocyte Activation Assay - LuSens

In at least two independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed.b

Negative

Dendritic Cell Line Activation Assay Human

Cell Line Activation Test (h-CLAT)

Not determined

Not determined

aNegative depletions were considered to be “zero” for calculation of mean peptide depletion.

bFor details of evaluation criteria see section3.10.2.

 

Based on the results summarized in Table 2 and applying the evaluation criteria, 3 -Methyl-1 -butanol is not peptide reactive and does not activate keratinocytes. 3-Methyl-1-butanol is predicted not to be a skin sensitizer.

There is a study available assessing the skin sensitization potential of 3 -methylbutan-1 -ol in humas and further results for other members of the category “pentanols”.

In the human maximization study (Kligman 1976), 25 human volunteers received an application of 8% 3 -methylbutan-1 -ol in a 2.5 % aqueous sodium lauryl sulfate solution. The substance was applied via an occlusive patch test to the same site on the volar forearm or back of all subjects for five alternate-day 48 hour periods. Evaluation of the skin sites revealed that none of the 25 individuals showed any signs of sensitization. The study was adequately documented and is considered the most reliable publication on human data, but the number of test subjects was still rather low.

The negative result is supported by recent in vitro guideline studies on pentanol. A detailled read across justification is attached in IUCLID chapter 13 and in the category object for pentanols. The in vitro assays address key events of the adverse outcome pathway of skin sensitization. In the direct peptide reactivity assay, pentanol was incubated for 24h with either cysteine (C-peptide) or lysine reach (K-peptide) synthetic peptides. C-peptide depletion was -1.46%, and K-peptide depletion was 2.88% compared to the vehicle control. Since negative values are considered as "zero", the average peptide depletion was 1.44%. All values below 4.65% are assessed as negative in this assay. Consequently, pentanol shows minimal or no chemical reactivity in this assay. The ability of pentanol to activate keratinocytes was evaluated in the LuSens assay. Pentanol was incubated with human transgenic HaCat cells for 48h, and activation of the antioxidant response element was measured as luminescence. Concentrations that reduced viability to below 70% were not assessed. As a result, luciferase activity was not increased by at least a factor of 1.5 above control values. From this is has to be concluded that the test substance does not have a keratinocyte activating potential. Because the results were clearly negative for the first two in vitro assays, pentanol is predicted not to be as skin sensitizier. No additional test for dendritic cell activation is required.

There are also a few publications available assessing the skin sensitization potential of the pentanol or its isomers in humans. The value of these three case reports is fairly limited. The very few reports on positive patch test responses are most likely due to cross-reactivity (proven in 2 cases), and the purity of the test material is questionable. In an epicutaneous patch test (Fregert 1963), one dermatitiis patient reacted to pentan-1 -ol at a concentration of 10 %, but the chromatogram of the test substance showed 11 additional peaks, so the cause for the reaction is unclear. In another publication, four patients also exhibiting dermatitis were tested positive for pentan-1 -ol (Fregert 1969). A further test person, who showed positive skin reactions towards hair lotions, was exposed to a series of lower aliphatic alcohols and did not react to pentan-1 -ol in an epicutaneous patch test (Ludwig 1977). It should be noted that all patients with positive reactions to pentanol also reacted to all other tested lower primary alcohols. From the provided anamnesis data it can be concluded that pentanol was an unlikely inducer of the sensitzisation. Definite cross sensitization was observed in the publication by Stotts (1977). One patient had previously been sensitized against ethanol due to his voluntary participation in a human patch testing procedure. The other person was sensitized against acetaldehyde while undergoing testing for the maximum non-irritant concentration of this substance. During subsequent patch testing, both also reacted to pentanol, but also to all other tested primary alcohols.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data are considered reliable and suitable for classification purposes under Regulation (EC) No 1272/2008 (CLP).

As a result, the substance does not need to be classified for skin sensitisation under Regulation (EC) No 1272/2008.