Chlorbutanol

Endpoint summary

Description of key information

Two in vitro studies have been performed to analyse the sensitising potential of Chlorobutanol. No relevant sensitisation has been observed under test conditions, thus Chlorobutanol is considered not skin sensitising

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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Due to a HPLC error causing a delayed start of the sequence, the calibration sam-ples had to be moved from the beginning to the end of the analysis sequence to ensure measurement of the peptide samples in a timely manner. The deviation was assessed and signed by the study director on 30. Jan. 2018.

Deviations:

yes

Remarks:

see remarks

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The direct peptide reactivity assay (DPRA) is an in chemico assay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential. This study was performed in order to estimate the skin sensitisation potential of Chlorobutanol Hemihydrate using a peptide model. To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item was measured using HPLC. The assay was used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible.

Run / experiment:

other: Cysteine

Parameter:

other: Cys-peptide depletion [%]

Value:

2.77

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Lysine

Parameter:

other: Lys-Peptide depletion [%]

Value:

1.75

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: mean (Cysteine and Lysine)

Parameter:

other: Mean peptide depletion [%]

Value:

2.26

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Table: Calculated peptide depletion values for the Lys-Peptide

Sample name	Depletion [%]
	Single	Mean	Standard Deviation
Positive control Rep. 1	28.07	34.12	8.69
Positive control Rep. 2	30.21
Positive control Rep. 3	44.08
Test item Rep. 1	0.00	1.75	3.04
Test item Rep. 2	0.00
Test item Rep. 3	5.26

 

Table: Calculated peptide depletion values for the Cys-Peptide

Sample name	Depletion [%]
	Single	Mean	Standard Deviation
Positive control Rep. 1	83.72	84.28	0.75
Positive control Rep. 2	85.13
Positive control Rep. 3	83.99
Test item Rep. 1	0.00	2.77	2.43
Test item Rep. 2	3.75
Test item Rep. 3	4.54

 

Interpretation of results:

GHS criteria not met

Conclusions:

All acceptance criteria were fulfilled, therefore the test was considered valid. The DPRA prediction for the test item Chlorobutanol Hemihydrate was negative with reactivity class minimal 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:

The study was performed in order to evaluate the reactivity of the test item Chlorobutanol Hemihydrate towards cysteine (Cys-) and lysine (Lys-) containing peptides. A 100 mM test item in acetonitrile was incubated 24 ± 2 h at 25 °C together with cysteine and lysine peptides, 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 in parallel. The peptide depletion values after incubation are: 

Cys-peptide depletion [%]	Lys-Peptide depletion [%]	Mean peptide depletion [%]
2.77	1.75	2.26

 According to the cysteine 1:10/lysine 1:50 prediction model, the DPRA predicition is “negative” with minimal reactivity.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09. Oct. 2017 - 10. Nov. 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

This in vitro study is performed to assess the sensitizing potential of the test item Chlorobutanol Hemihydrate by using the genetically modified keratinocyte cell-line “LuSens” (LuSens, Bauch et al. 2012). The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay). The assay differs in some points from the OECD guideline.The LuSens test employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with test substances. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signalling pathway (Ade et al. 2009, Natsch 2012, Natsch & Emter 2008, Vandebriel et al. 2010). In order to conclude on the skin sensitization potential of the test substance, a LuSens test, comprising at least two, but a maximum of three inde-pendent and valid experiments will be performed. The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible.

Key result

Run / experiment:

other: Experiment I:

Parameter:

other: Induction of Luciferase

Value:

0.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

for detailed information see table I

Key result

Run / experiment:

other: Experiment II

Parameter:

other: Induction of Luciferase

Value:

0.85

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

for detailed information see table II

table I: results of experiment I

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.05	5.38	100.0	4.09	4.09
Growth Control	-	1.1	0.07	6.22	133.1	6.70	5.04
Negative Control	5000	1.0	0.03	3.15	103.3	6.81	6.60
Positive Control	120	5.2	0.35	6.72	85.1	4.64	5.45
Test item	269	1.0	0.04	3.52	103.6	3.49	3.36
Test item	323	0.9	0.04	4.36	101.2	2.05	2.03
Test item	388	0.8	0.03	3.68	99.4	0.82	0.82
Test item	465	0.9	0.02	2.09	96.0	2.97	3.09
Test item	558	0.8	0.01	1.36	92.8	2.47	2.67
Test item	670	0.8	0.01	1.37	88.8	1.91	2.16
Test item	804	0.8	0.02	1.93	87.4	0.52	0.59
Test item	965	0.8	0.02	2.30	88.4	2.36	2.67
Test item	1157	0.8	0.04	4.96	85.7	2.81	3.28
Test item	1389	0.8	0.09	11.44	78.6	6.89	8.76
Test item	1667	0.8	0.03	3.31	74.8	11.91	15.93
Test item	2000	0.8*	0.02	3.12	56.3	2.79	4.95

* = Due to cytotoxicity value was not used for evaluation

table II: results of experiment II

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.10	9.63	100.0	3.62	3.62
Growth Control	-	1.0	0.06	5.84	139.9	5.84	4.18
Negative Control	5000	1.0	0.05	5.20	111.5	6.75	6.05
Positive Control	120	4.2	0.13	3.05	97.0	3.12	3.21
Test item	269	1.0	0.03	3.32	110.5	3.49	3.16
Test item	323	0.9	0.02	1.92	106.4	5.37	5.04
Test item	388	0.9	0.01	1.31	102.8	4.77	4.64
Test item	465	0.9	0.06	6.63	99.2	5.66	5.71
Test item	558	0.8	0.02	1.86	90.8	2.67	2.95
Test item	670	0.8	0.02	2.89	90.9	2.05	2.26
Test item	804	0.8	0.05	5.71	92.7	1.50	1.62
Test item	965	0.8	0.04	5.02	88.1	0.42	0.47
Test item	1157	0.8	0.04	4.29	85.7	3.39	3.95
Test item	1389	0.8	0.08	10.14	79.6	2.32	2.92
Test item	1667	0.9	0.08	8.97	75.0	3.89	5.19
Test item	2000	1.0*	0.11	10.89	65.6	1.38	2.11

* = Due to cytotoxicity value was not used for evaluation

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, the test item, Chlorobutanol Hemihydrate, was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor (no sensitizing potential).

Executive summary:

This in vitro study evaluates the potential of the test item Chlorobutanol Hemihydrate to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated ap-proach to testing and assessment. The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens As-say). The assay differs in some points from the OECD guideline. The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined. In the experiments, the highest nominal applied concentration (2000 µM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2 ) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments. DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA  (120 µM) as positive control. No substantial and reproducible dose dependent increase in luciferase induction above 1.5 fold was observed in both experiments up to the maximal tested concentration of the test item.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available