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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08-11-2017 - 11-20-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
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. 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.
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.

In chemico test system

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”.

Results and discussion

Positive control results:
see section "Any other information on results incl. tables"

In vitro / in chemico

Resultsopen allclose all
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.