Butyl O-butyryllactate

Endpoint summary

Description of key information

Study performed to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2018 to 15 May 2018

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:

04 Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

This is a Non in vivo test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in vivo studies on these types of raw materials.

Specific details on test material used for the study:

Appearance: Colourless liquid
Purity: 99.77%
Test item storage: At room temperature

Details on the study design:

MATERIALS AND METHODS:
Test Item and Reference Item:
Test Item (Butyl Butyryl Lactate):

Identification: Butyl Butyryl Lactate
Appearance: Colourless liquid
Batch: 202432
Purity/Composition: 99.77%
Test item storage: At room temperature
Stable under storage conditions until: 16 September 2018 (expiry date) Additional information
Test Facility Test Item Number: 209414/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC, synonym or trade name: Butyl O-butyryl lactate
CAS number: 7492-70-8
EC number: 231-326-3
Molecular formula: C11H20O4
Molecular weight: 216.28 g/mol

Reference Item (Positive Control Cinnamic Aldehyde)
Identification: Cinnamic aldehyde
Test Facility Test Item Number: RS473/A
Appearance: Yellow liquid
CAS Number: 104-55-2
Molecular Formula: C9H8O
Molecular Weight: 132.16 g/mol
Batch: MKBP1014V
Purity: 98.4%
Test item storage: In the refrigerator (2-8°C)

Stable under storage conditions until: 31 May 2018
Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
Purity/composition correction factor: Yes
For Certificate of Analysis see Appendix 6.

Test Item Characterization:
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. A Certificate of Analysis was provided to the Test Facility and is presented in Appendix 6.

Reserve Samples:
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

Test Item Inventory and Disposition:
Records of the receipt, distribution, and storage of test item were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

Dose Formulation and Analysis:
Preparation of Test Item:
No correction for the purity/composition of the test item was performed.

Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), dimethylsulfoxide (DMSO):ACN (1:9, v/v), isopropanol, methanol (MeOH) and ethanol (EtOH).

Test item stock solutions were prepared freshly for each reactivity assay.

For both the cysteine and lysine reactivity assay 36.08 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1668 µL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

Any residual volumes were discarded.

Test system: Synthetic peptides containing cysteine (SPCC) Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.

Rationale: Recommended test system in the international OECD guideline for DPRA studies.
Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Batch: See Appendix 7 for detailed information.
Storage: The peptides were stored in the freezer (≤-15°C) for a maximum of 6 months.

Reagents:
Acetonitrile (ACN): HPLC grade, Fisher Chemicals, Loughborough, England
Ammonium acetate: Fractopur, Merck, Darmstadt, Germany
Ammonium hydroxide: 25%, Merck
Dimethylsulfoxide (DMSO): Seccosolv, Merck
Disodium hydrogen phosphate (Na2HPO4·12H2O): Emsure, Merck
Ethanol (EtOH): LiChrosolv, Merck
Isopropanol: LiChrosolv, Merck
Methanol (MeOH): BioSolve, ULC/MS
MilliQ-water (MQ): Tap water purified by reversed osmosis and subsequently passed over activated carbon and ion exchange cartridges; Millipore, Bedford, MA, USA
Sodium dihydrogenphosphate dehydrate (NaH2PO4·H2O): Emsure, Merck
Trifluoroacetic acid (TFA): >99%, Sigma Aldrich, Zwijndrecht, The Netherlands

Experimental Design:
Preparation of Solutions for Cysteine Reactivity Assay:
Synthetic Peptide: Containing Cysteine (SPCC) Stock Solution
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

SPCC Reference Control Solutions:
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.

SPCC Calibration Curve:
A SPCC calibration curve was prepared as described in the table below:

Preparation of SPCC Calibration Curve:

SPCC calibration solutions SPCC concentration (mM) Preparation
STDcys1 0.534 1600 µL stock solution of 0.667 mM SPCC + 400 µL ACN
STDcys2 0.267 1 mL STDcys1 + 1 mL STDcys7
STDcys3 0.133 1 mL STDcys2 + 1 mL STDcys7
STDcys4 0.067 1 mL STDcys3 + 1 mL STDcys7
STDcys5 0.033 1 mL STDcys4 + 1 mL STDcys7
STDcys6 0.017 1 mL STDcys5 + 1 mL STDcys7
STDcys7 0 8 mL phosphate buffer (pH 7.5) + 2 mL ACN


Co-elution Control, Test Item and Positive Control Samples:
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table below.

Preparation of Co-elution Control, Test Item and Positive Control Samples:

Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CCcys-209414/A 750 µL Phosphate buffer pH 7.5
200 µL ACN
50 µL 209414/A test solution (100 mM)

Cinnamic aldehyde (PC) 3 PCcys-1 to PCcys-3 750 µL Stock solution of 0.667 mM SPCC
200 µL ACN
50 µL Cinnamic aldehyde solution
(100 mM in ACN)

Test item 209414/A 3 209414/A-cys-1 to 750 µL Stock solution of 0.667 mM SPCC
209414/A-cys-3 200 µL ACN
50 µL 209414/A test solution (100 mM)

Sample Incubations:
After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.5 hours and 25 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC-PDA analysis the samples were visually inspected for precipitation. The samples that showed a phase separation were centrifuged (at 400 g) for 5 minutes at room temperature.

HPLC-PDA Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
• LC Column oven 300 (Thermo Scientific)
• Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
• Column Oven #151006 (Grace, Worms, Germany)
• Surveyor PDA detector (Thermo Scientific)
All samples were analyzed according to the HPLC-PDA method presented in Table 1 (Appendix 1). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 (Appendix 1).

ACCEPTABILITY CRITERIA:
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.

The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Positive control results:

The results of the positive control cinnamic aldehyde are presented in Table 7 (Appendix 3). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 76.6% ± 1.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Key result

Parameter:

other: SPCC depletion

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Parameter:

other: SPCL depletion

Value:

0.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

RESULTS

Solubility Assessment of the Test Item

At a concentration of 100 mM, Butyl Butyryl Lactate was soluble in ACN, DMSO:ACN (1:9, v/v), isopropanol, EtOH and MeOH.

Solubility of the 100 mM test item solution prepared in ACN, DMSO:ACN (1:9, v/v),isopropanol, EtOH and MeOH was investigated in the SPCC assay buffer by mixing 50 µL ofthe 100 mM test item solution with 750 µL phosphate buffer pH 7.5 and 200 µL ACNfollowed by vortex mixing. When the test item stock solution was prepared in ACN orDMSO:ACN (1:9, v/v), the test item was soluble in the phosphate buffer solution, however,when the test item stock solution was prepared in isopropanol, EtOH or MeOH, the test item was not soluble in the phosphate buffer solution.

Solubility of the 100 mM test item solution prepared in ACN, DMSO:ACN (1:9, v/v), isopropanol, EtOH and MeOH was investigated in the SPCL assay buffer by mixing 250 µL of the 100 mM test item solution with ammonium acetate buffer pH 10.2 followed by vortex mixing. When the test item stock solution was prepared in ACN or DMSO:ACN (1:9, v/v), formation of a phase separation in the form of oily droplets was observed. When the test item stock solution was prepared in isopropanol, EtOH or MeOH, the test item was not soluble in

the ammonium acetate buffer solution.

As ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in this DPRA study.

Cysteine Reactivity Assay

The reactivity of Butyl Butyryl Lactate towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 24.5 hours of

incubation at 25±2.5°C. Representative chromatograms of CCcys-209414/A and 209414/A-cys samples are presented in Appendix 4. An overview of the retention time at

220 nm and peak areas at 220 nm and 258 nm are presented in Table 3 (Appendix 3).

Acceptability of the Cysteine Reactivity Assay

The SPCC standard calibration curve is presented in Figure 1 (Appendix 2). The correlationcoefficient (r2) of the SPCC standard calibration curve was 0.997. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 4 (Appendix 3).The mean peptide concentration of Reference Controls A was 0.519 ± 0.002 mM while the mean peptide concentration of Reference Controls C was 0.509 ± 0.007 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.

The SPCC peak areas for Reference controls B and C are presented in Table 5 (Appendix 3). The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and

C was 2.0%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCC A220/A258 area ratios of Reference controls A, B and C are presented in Table 6 (Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 19.51. The mean A220/A258 ratio ± 10% range was 17.56-21.46. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 7 (Appendix 3). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehydewas 76.6% ± 1.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Results Cysteine Reactivity Assay for the Test Item

Preparation of a 100 mM Butyl Butyryl Lactate stock solution in ACN showed that the testitem was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples.

The results of the cysteine reactivity assay for the test item are presented in Table 8 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCC (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCC. For the 209414/A-cys samples, the mean SPCC A220/A258 area ratio was

19.63. Since this was within the 17.56-21.46 range, this again indicated that there was no co-elution of the test item with SPCC.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 0.0% ± 0.0%.

Lysine Reactivity Assay

The reactivity of Butyl Butyryl Lactate towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 25 hours of incubation at 25±2.5°C. Representative chromatograms of CClys-209414/A and 209414/A- lys samples are presented in Appendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 9 (Appendix 3).

Acceptability of the Lysine Reactivity Assay

The SPCL standard calibration curve is presented in Figure 2 (Appendix 2). The correlationcoefficient (r2) of the SPCL standard calibration curve was 0.9995. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 10 (Appendix 3). The mean peptide concentration of Reference Controls A was 0.490 ± 0.014 mM while the mean peptide concentration of Reference Controls C was0.505 ± 0.023 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

The SPCL peak areas for Reference controls B and C are presented in Table 11 (Appendix 3). The CV of the peptide areas for the nine Reference Controls B and C was 4.2%. This was

within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCL A220/A258 area ratios of Reference controls A, B and C are presented in Table 12 (Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 18.55. The mean A220/A258 ratio ± 10% range was 16.69-20.40. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 13 (Appendix 3). The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference

Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehydewas 50.7% ± 3.0%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Results Lysine Reactivity Assay for the Test Item

Preparation of a 100 mM Butyl Butyryl Lactate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation as well as after incubation a phase separation was observed in the CC and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

The results of the lysine reactivity assay for the test item are presented in Table 14 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCL (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCL. For the 209414/A-lys samples, the mean SPCL A220/A258 area ratio was 18.28. Since this was within the 16.69-20.40 range, this again indicated that there was no co-elution of the test item with SPCL.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of ReferenceControls C. The mean Percent SPCL Depletion for the Test Item was 0.8% ± 1.4%.

DPRA Prediction and Reactivity Classification

Upon preparation as well as after incubation of the SPCC test item samples, no precipitate or phase separation was observed in any of the samples. Upon preparation as well as after

incubation of the SPCL test item samples, a phase separation was observed.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine reactivity assay the test item showed 0.0% SPCC depletion while in the lysine reactivity assay the test item showed 0.8% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.4% and as a result the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forthe Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Butyl Butyryl Lactate	0.0%	±0.0%	0.8%	±1.4%	0.4%	Negative: No or minimal reactivity

SD = Standard Deviation.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, this DPRA test is valid. Butyl Butyryl Lactate was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since phase separation was observed after the incubation period for SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Executive summary:

In this guideline (OECD 442C) study, performed with GLP certification, the test substance Butyl Butryl Lactate (EC 231-326-3) was found to not be classified as a skin sensitiser under Regulation EC 1272/2008.

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

QSAR: DEREK NEXUS

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
DEREK NEXUS v 6.0.1.

2. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Chemical name: Butyl Butyryl Lactate
CAS Number 7492-70-8
Molecular weight: 216.28
Molecular formula C11H20O4

3. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitization based on the presence of molecular substructures. LHASA (see Appendix I) has inserted validation comments for the skin sensitization alerts.

4. APPLICABILITY DOMAIN
See QMRF

5. ADEQUACY OF THE RESULT
See QMRF

Qualifier:

no guideline required

Principles of method if other than guideline:

DEREK NEXUS contains 90 alerts for skin sensitisation, together with reasoning rules encoding physicochemical descriptors. In addition to a prediction of skin sensitisation potency for alerting query compounds, Derek evaluates potentially misclassified and unclassified features in cpompounds that do jot activate skin sensitsation alerts or examples.

GLP compliance:

no

Remarks:

QSAR data

Justification for non-LLNA method:

QSAR data used as part of an in vitro testing strategy.

Parameter:

other: Structural alerts

Value:

0

Parameter:

other: Unclassified features

Value:

0

Parameter:

other: misclassified features

Value:

0

Other effects / acceptance of results:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer.

Interpretation of results:

GHS criteria not met

Conclusions:

Butyl Butyryl Lactate is predicted to be not sensitizing to the skin.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 2018 to 14 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™

Version / remarks:

Adopted March, 2018

Deviations:

no

Principles of method if other than guideline:

Test System
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Rationale
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD 442D).

Cell Culture:
Basic medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).

Maintenance medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).

Exposure medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 86 – 99%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 – 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Subculturing:
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

Experimental Design:
Plating of Cells:
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+13 in experiment 1 and P+11 in experiment 2.

Treatment of Cells:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0oC in the presence of 5% CO2. In total 2 experiments were performed.

Luciferase Activity Measurement:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This is a Non in-vivo test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in-vivo studies on these types of raw materials.

Specific details on test material used for the study:

Identification: Butyl Butyryl Lactate
Appearance: Colourless liquid
Purity/Composition: 99.77%, assumed 100% for test
Test item storage: At room temperature

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.24 and the EC1.5 was 70 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 1.88 and the EC1.5 was 107 µM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.03

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.05

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Butyl Butyryl Lactate was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. An overview of the viability and luciferase activity induction is summarized inTable1andFigure2-3. The results of the positive control are summarized in Table 2 and Figure 4-5. An overview of EC_1.5, I_max, IC₃₀and IC₅₀ values is given in Table 3. The individual raw data are presented in Appendix 3 and Appendix 4. The historical control data are presented in Appendix 5.

Two independent experiments were performed. The cells were in these experiments incubated withButyl Butyryl Lactatein a concentration range of 0.98 – 2000 µM (2-fold dilution steps) for 48 hours± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

Experiment 1

·          No precipitation was observed at the start and end of the incubation period in the 96-well plates.

·          Butyl Butyryl Lactate showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC₃₀ and IC₅₀ values could be calculated.

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Butyl Butyryl Lactate. The I_maxwas 1.03 and therefore no EC_1.5 could be calculated.

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_maxwas 2.24 and the EC_1.5 70 µM. 

Experiment 2

·          No precipitation was observed at the start and end of the incubation period in the 96-well plates.

·          Butyl Butyryl Lactate showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC₃₀ and IC₅₀ values could be calculated.

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Butyl Butyryl Lactate. The I_max was 1.05 and therefore no EC_1.5 could be calculated.

·          The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The I_max was 1.88 and the EC_1.5 107 µM. 

Both tests passed the acceptance criteria:

·          The luciferase activity induction obtained with the positive control,Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. 

·           The EC_1.5 should be within two standard deviations of the historical mean (70 µM and 107 µM in experiment 1 and 2, respectively). A dose response was observed in both experiments and the induction at 250 µM was higher than 2-fold in experiment 1 only (2.24-fold and 1.88-fold in experiment 1 and 2, respectively).

·          Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (10% and 7.3% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Butyl Butyryl Lactate is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of Butyl Butyryl Lactateto activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay.

 

The study procedures described in this report were based on the most recent OECD guideline.

 

The Butyl Butyryl Lactate was a colourless liquid. Butyl Butyryl Lactate was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). The highest test concentration was thehighest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

 

Both experiments passed the acceptance criteria:

·          The luciferase activity induction obtained with the positive control,Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. 

·           The EC_1.5 was within two standard deviations of the historical mean (actual values 70 µM and 107 µM in experiment 1 and 2, respectively). A dose response was observed in both experiments and the induction at 250 µM was higher than 2-fold in experiment 1 only (2.24-fold and 1.88-fold in experiment 1 and 2, respectively).

·          Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (10% and 7.3% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

 

Butyl Butyryl Lactate showed no toxicity (no IC₃₀ and IC₅₀ value) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.03-fold and 1.05-fold in experiment 1 and 2 respectively. Butyl Butyryl Lactate is classified as negative in the KeratinoSens™ assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000µM.

 

In conclusion, Butyl Butyryl Lactate is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The test substance was shown to be non-sensitising in the DPRA and Keratosens in vitro tests and the DEREK in chemico assessment