Pentan-1-ol

Endpoint summary

Description of key information

Pentan-1-ol is not peptide reactive and does not activate keratinocytes. Consequently, Pentan-1 -ol is predicted not to be a skin sensitizer.

This is supported by data on 3 -methylbutan-1 -ol, which did not cause sensitization in humans.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For justification of read across please refer to section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Tk287_20151217
- Expiration date of the lot/batch: 17.12.2016
- Purity test date: 20.04.2016
- Purity: 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: guaranteed by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: soluble, no analysis performed because the dissolved test substance was used shortly after preparation

Details on the study design:

The test substance is incubated with synthetic peptides for 24 hours at room temperature in the dark. The remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm.
The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.

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 RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

Concentrations:
C-peptide: 5mM test substance, 0.5mM peptide
K-peptide: 25mM test substance, 0.5mM peptide
Test substance preparations were prepared on a weight per volume basis within 4 hours of the start of the experiment.
No. of replicates: 3

Vehicle control: acetonitrile
Reason for vehicle: solubility
Positive control: Ethylene glycol dimethacrylate (50mM in acetonitrile)

Key result

Parameter:

other: Peptide depletion

Value:

1.44

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: C-peptide depletion

Value:

-1.46

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: K-peptide depletion

Value:

2.88

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

The standard calibration curve should have an r² >0.99.
The vehicle control samples should be 0.50 mM +/- 0.05mM.
The CV of the nine vehicle controls should be < 15%.
The variability between these samples should be acceptably low (SD <14.9% for % cysteine depletion and <11.6% for % lysine depletion).
The positive control should cause depletion of both peptides comparable to historic data.

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For justification of read across please refer to section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Tk287_20151217
- Expiration date of the lot/batch: 17.12.2016
- Purity test date: 20.04.2016
- Purity: 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: guaranteed by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: the substance was soluble in 1% and 4% DMSO in cell culture medium (visual inspection). The stability was not analysed, because the test substance preparations were used shortly after preparation.

Details on the study design:

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. Viability was checked via MTT and should exceed 70%. A total of 2 valid experiments were performed with three replicates each.

Dose setting:
In a pre-test the concentration that reduces viability by 25% was determined to be 363µg/mL
The following concentrations were used in both experiments: 175, 210, 252, 302, 363, 435, 523, 627µg/mL.

Cell Line:
Human transgenic keratinocyte cell line derived from HaCaT cells
Culture medium: DMEM + FBS

Controls:
Negative control: DL-lactoc acid (CAS 50-21-5, 450µg/mL in 1% DMSO
Positive control: Ethylene glycol dimethacrylate (DAS 97-90-5), 18µg/mL in 1% DMSO
Vehicle control: 1% DMSO
Blank control: Culture medium w/out cells
Basal control: Culture medium w/ cells

Key result

Parameter:

other: Fold Luciferase induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Acceptance criteria

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 negative control <1.5 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 <1.5 as compared to the solvent control.
In addition, positive, negative and vehicle control data should lie within the range of the historic data.

Concentration (test substance) µg/mL	fold induction	1st experiment rel. viability [%]               t-test			Concentration (test substance) µg/mL	fold induction	2nd experiment rel. viability [%]               t-test
	mean	mean	p-value	markers		mean	mean	p-value	markers
175	1.09	81.1	0.207	n.s.	175	0.94	82.4	0.141	n.s.
210	1.22	73.9	0.071	n.s.	210	0.91	77.1	0.265	n.s.
252	1.25	75.7	0.002	**	252	1.00	72.5	0.489	n.s.
302	1.35	75.3	0.013	*	302	1.11	69.0	0.302	n.s.
363	1.23	73.4	0.021	*	363	1.00	72.5	0.489	n.s.
435	1.43	71.2	0.001	**	435	1.11	74.5	0.108	n.s.
523	1.36	61.6	0.000	**	523	1.20	60.7	0.200	n.s.
627	1.50	52.8	0.000	**	627	1.20	46.1	0.041	*
VC	1.00	100.0	-	-	VC	1.00	100.0	-	-
EGDMA (18 µg/mL)	6.64	108.6	0.000	**	EGDMA (18 µg/mL)	7.61	121.3	0.000	**
LA (450 µg/mL)	0.76	103.3	0.000	**	LA (450 µg/mL)	1.01	104.8	0.441	n.s.

LA: lactonic acid

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitzing potential of pentanol was evaluated using several in vitro assays that 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.

A few publications are available assessing the skin sensitization potential of the pentanol or its isomers in humans. The value of the three case reports is fairly limited. The most reliable study is the human maximisation test with 3 -methylbutan-1 -ol in which no sensitization was caused by the test substance. But also in this case the number of test subjects is far below what would be required for a reliable negative result. 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. Please refer to IUCLID5, section 7.10.4 for details.

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. A detailled read across justification is attached in IUCLID chapter 13. 

 

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 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.

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-butanolthe 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 -butanoltowards 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 following results were obtained in the DPRA:

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 modelit 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-butanol was 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 summary, after 48 hours of exposure to test substance3 -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.

 3-Methyl-1-butanol is not peptide reactive and does not activate keratinocytes.3-Methyl-1-butanolis predictednot to be a skin sensitizer.

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.