Geranyl butyrate

Endpoint summary

Description of key information

Skin Irritation:

The dermal irritation potential of test article Geranyl butyrate (CAS No.- 106-29-6) was determined according to the OECD 439 test guideline followed for this study. The Mean % tissue viability compared to negative control (n=3) of the test substance Geranyl butyrate was determined to be 102.5%. Thus, substance Geranyl butyrate (CAS No: 106-29-6) was considered to be not irritating to the human skin.

Eye Irritation:

The ocular irritation potential of test article Geranyl butyrate (CAS No.106-29-6) was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance Geranyl butyrate was determined to be 107.7%. Thus, substance Geranyl butyrate (CAS No.106-29-6) is considered to be "not irritating" to the human eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 05, 2017 to July 17, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Principles of method if other than guideline:

The purpose of this study was to assess potential for the test articles to be dermal irritants. The dermal irritation potential of test article Geranyl butyrate (CAS No.106-29-6) may be predicted by measurement of their cytotoxic effect, as reflected in the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiDerm™ model (MatTek Corp., Ashland, MA).

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test material identity: Geranyl butyrate
- Source: Otto Chemie Pvt Ltd.
- Batch No.of test material: 0027
- Manufacture date: 05/08/2016
- Expiration date of the lot/batch: 05/08/2021
- Purity: 95.05%
- Molecular formula: C14H24O2
- Molecular weight: 224.34
- Appearance: Colorless to pale yellow liquid

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: No data available
- Solubility and stability of the test substance in the solvent/vehicle: No data available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test articles is tested as provided (neat).
- Preliminary purification step (if any): No data available
- Final dilution of a dissolved solid, stock liquid or gel: No data available
- Final preparation of a solid: No data available

FORM AS APPLIED IN THE TEST: Liquid

OTHER SPECIFICS: No data available

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ 3-dimensional human tissues used in this study

Source strain:

other: Not applicable

Details on animal used as source of test system:

- Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

Test System Identification
All of the EpiDerm™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues are included in this report. Tissue plates were appropriately labeled with study information.

Justification for test system used:

The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, Ashland, MA) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.

Vehicle:

unchanged (no vehicle)

Details on test system:

The tissues were exposed to the test article Geranyl butyrate neat (undiluted) on June 28, 2017 (Run 1 of 1). EpiDerm™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.

MTT and Color Pre-tests
Pretesting for MTT auto-reduction and coloring was not performed for this study but was based on the results obtained from another study (CYP1690_R1b).

MTT Assay
Following the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 2 hours, 57 minute and 25 second MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was approximately 2 hours 04 minutes and 11 seconds with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean negative control tissues.

Evaluation of Test Article in the Cell Models:
1. Cell system: Upon receipt, the MatTek EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight at ~37°C, 5% CO2 in a humidified incubator.

2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test articles are applied topically to tissues by pipette. Tissues were exposed to controls and the test articles for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.

a) Controls
30 µL of negative control DPBS, positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.

b) Test Articles
30 µL of the geranyl butyrate was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.

3. Post-exposure treatment
After the 1 hour exposure, the tissues were rinsed 20 to 25 times with 1 mL of DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for either 25 hours, 38 minutes and 23 seconds or for 24 hours, 10 minutes and 09 seconds (as there were numerous tissues, they had to be broken down into 2 sets to complete dosing in a timely manner). After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 17 hours, 03 minutes and 34 seconds prior to performing the MTT assay, for a total of an approximately 42 hour post-exposure incubation.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The EpiDerm™ 3 dimensional human tissue model
- Tissue Lot number(s): 26459
- Date of initiation of testing: 6/08/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Twice

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL MTT medium (1.0 mg/mL).
- Incubation time: After 2 hours, 57 minute and 25 second MTT incubation
- Spectrophotometer: Synergy H4 spectrophotometer
- Wavelength: 570 nm
- Filter: No data
- Filter bandwidth: No data
- Linear OD range of spectrophotometer: No data

NUMBER OF REPLICATE TISSUES: 3

CALCULATIONS and STATISTICAL METHODS
All data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows:

MTT Assay
Blanks:
·        The optical density (OD) mean from all replicates for each plate (ODblank).

Negative Controls (NC):
·        The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
·        The OD mean per NC tissue was calculated.
·        The mean OD for all tissues corresponds to 100% viability.
·        The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.

Positive Control (PC):
·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
·        The OD mean per PC tissue was calculated.
·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.

Tested compound :
·        Calculate the blank corrected value ODTT= ODTTraw– ODblank.
·        The OD mean per tissue was calculated.
·        The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100.
·        The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues.
·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.

Data Correction Procedure for MTT Interfering Compounds (if applicable)
True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).
ODtvt= optical density of treated viable tissue
ODkt= optical density of killed tissues
ODtkt= optical density of treated killed tissue
ODukt= optical density of untreated killed tissue (NC treated tissue)

Data Correction Procedure for Colored Compounds (if applicable)
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt– ODvt.
ODtvt= optical density of treated viable tissue incubated in MTT media
ODvt= optical density of viable tissues incubated in media alone
 
- Evaluation of data
The results of the assay was evaluated and compared to negative control.

Table: Criteria for in vitro Interpretation:
In VitroResults In VivoPrediction
Mean tissue viability ≤50% Irritant (I), R38
Mean tissue viability >50% Non-irritant (NI)

- Assay quality controls
- Negative Controls (NC)
The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.
 
- Positive Controls (PC)
5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.
 
- Standard Deviation (SD)
The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): neat

VEHICLE (Not used)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% solution of sodium dodecyl sulfate

Duration of treatment / exposure:

The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

For a total of an approximately 42 hour post-exposure incubation.

Number of replicates:

3 tissues were used for test compound and control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Run 1

Value:

102.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 1.3 passing the acceptance criteria. The Standard Deviation, initially caused the test articles to not pass acceptance criteria (reported as 36.9 which is >18%) but after removing a single outlier, it has passed the acceptance criteria.

Interpretation of results:

other: not irritating

Conclusions:

The dermal irritation potential of test article Geranyl butyrate (CAS No.- 106-29-6) was determined according to the OECD 439 test guideline followed for this study. The Mean % tissue viability compared to negative control (n=3) of the test substance Geranyl butyrate was determined to be 102.5%. Thus, substance Geranyl butyrate (CAS No: 106-29-6) was considered to be not irritating to the human skin.

Executive summary:

The dermal irritation potential of test article Geranyl butyrate (CAS No.- 106-29-6) was determined according to the OECD 439 test guideline followed for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to test articls and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 1.3 passing the acceptance criteria.

The Mean % tissue viability compared to negative control (n=3) of the test substance Geranyl butyrate was determined to be 102.5%.

Hence, under the experimental test conditions it was concluded that test substance Geranyl butyrate (CAS No.- 106-29-6) was considered to be not irritating to the skin of humans and being classified as “Not Classified'' as per CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 05, 2017 to July 12, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Principles of method if other than guideline:

The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article Geranyl butyrate (CAS No.106-29-6) may be predicted by measurement of its cytotoxic effect, as reflected in the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiOcular™ model (MatTek Corp., Ashland, MA).

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test material identity: Geranyl butyrate
- Source: Otto Chemie Pvt Ltd.
- Batch No.of test material: 0027
- Manufacture date: 05/08/2016
- Expiration date of the lot/batch: 05/08/2021
- Purity: 95.05%
- Molecular formula: C14H24O2
- Molecular weight: 224.34
- Appearance: Colorless to pale yellow liquid

RADIOLABELLING INFORMATION (Not applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: No data available
- Solubility and stability of the test substance in the solvent/vehicle: No data available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article tested as provided neat (undiluted).
- Preliminary purification step (if any): No data available
- Final dilution of a dissolved solid, stock liquid or gel: No data available
- Final preparation of a solid: No data available

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid

OTHER SPECIFICS: No data available

Species:

human

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

- Description of the cell system used:
The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.

- Test System Identification
All of the EpiOcular™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues is included in this report. Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.

- Justification of the test method and considerations regarding applicability
Human Corneal Epithelia (HCE) by MatTek, Inc.:
The test articles and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek Corporation (Ashland, MA). This model consists of normal human keratinocytes cultured on a permeable synthetic membrane at the air-liquid interface in a chemically defined medium. The cells form a stratified, squamous corneal epithelium resembling the corneal mucosa of the human eye. This model has been used with several common tests of cytotoxicity including MTT and interleukin 1-alpha (IL-1α). A growing body of evidence indicates that EpiOcular™ effectively provides a non-animal means to assess potential irritancy. The EpiOcular™ model closely mimics the human corneal mucosa and thus provides an important in vitro approach in the evaluation of ocular irritancy and toxicity. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).

Vehicle:

unchanged (no vehicle)

Controls:

other: See ''Remark" for Control Samples used in the study

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): neat (undiluted)

VEHICLE (no vehicle)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat

Duration of treatment / exposure:

Tissues were exposed for approximately 30 minutes for liquid test article and controls, at approximately 37°C, 5% CO2 in a humidified incubator.

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~2 hours for liquid test article and controls.

Number of animals or in vitro replicates:

3 tissues were used for test compound and control.

Details on study design:

- Details of the test procedure used
The tissues were exposed to the test article Geranyl butyrate neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 30 minutes for liquid test articles and controls, at approximately 37°C, 5% CO2 in a humidified incubator. After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~12 minutes for liquid test articles and controls. Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~2 hours for liquid test article and controls. Tissue viability was assessed by 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

- MTT Auto reduction and colouring assessment
MTT Pre-test
The test article was assessed for the potential to interfere with the assay. Approximately 50 µL of liquid test article Geranyl butyrate was added to 1 mL of MTT media (~1 mg/mL) and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 55 minutes and 02 seconds. 50 µL of ultrapure water was used as a negative control.

- Test Article Color Test
Approximately 50 µL of Geranyl butyrate was added to 1.0 mL of ultrapure water and 2.0 mL isopropanol and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 04 minutes and 35 seconds. Samples were then added to the wells of a clear 96-well plate and the plate was read on a BioTek Synergy H4 (or equivalent) plate reader set to 570 nm. Test articles that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions).

- MTT Assay
After the recovery period, the MTT assay was performed on run 1 tissues by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 2 hours 50 minutes and 14 seconds (liquids) of MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the tissues were rinsed twice with DPBS. The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction for liquid exposed tissues was overnight incubation (22 hours 17 minutes and 52 seconds) with a 20 minute 24 second shake the following morning. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissues.

- Evaluation of Test Article in the cell Models
1. Cell System:
Upon receipt, the MatTek EpiOcular™ tissue cultures were placed in 1.0 mL of fresh Maintenance medium (in a 6-well plate) for 55 minutes and 16 seconds. The tissues were not incubated overnight.

2. Control and Test Article Exposures:
20 µL of calcium and magnesium free DPBS was added to each tissue and the tissues placed back into the incubator for 31 minutes 00 seconds. The controls and the test article will be applied topically to tissues by pipette. Three tissues will be used per test compound and control.

a)Controls:
50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.

b)Test Article:
50 µL of Geranyl butyrate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.

3. Post exposure treatment:
After the exposure, the tissues were rinsed 20 to 25 times with ~1 mL of DPBS to remove test material. The apical surface was gently blotted with a cotton swab and cultures were immediately transferred to a 12-well plate containing 5 mL of media per well. Tissues exposed to liquid test articles (and the respective control) were incubated, submerged in the media for ~12 minutes at room temperature. Tissuses were then transferred to 6-well plates containing 1.0 mL fresh Maintenance medium per well and incubated for a post-exposure recovery period for 2 hours 01 minutes at approximately 37 degC, 5% CO2 in a humidified incubator.

- Doses of test chemical and control substances used
Test Article:
50 µL of Geranyl butyrate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.

Controls:
50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 30 minute exposure time.

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Tissues were exposed for approximately 30 minutes for liquid test article and controls, at approximately 37°C, 5% CO2 in a humidified incubator.
Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~2 hours for liquid test article and controls.

- Justification for the use of a different negative control than ultrapure H2O (Not applicable)

- Justification for the use of a different positive control than neat methyl acetate (Not applicable)


- Number of tissue replicates used per test chemical and controls: 3 tissues were used for test compound and control.

- Description of the method used to quantify MTT formazan
The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction for liquid exposed tissues was overnight incubation (22 hours 17 minutes and 52 seconds) with a 20 minute 24 second shake the following morning. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
Calculations and Statistical Methods
MTT Assay
Blanks:
·  The OD mean from all replicates for each plate (ODblank).

Negative Controls (NC):
·  The blank corrected value was calculated: ODNC= ODNCraw– ODblank.
·  The OD mean per NC tissue was calculated.
·  The mean OD for all tissues corresponds to 100% viability.
·  The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.

ODblank= optical density of blank samples (isopropanol alone).
ODNCraw= optical density negative control samples.
ODNC= optical density of negative control samples after background subtraction.

Positive Control (PC):
·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank.
·        The OD mean per PC tissue was calculated.
·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100.
·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues.
·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated.
ODPCraw= optical density positive control samples.
ODPC= optical density of positive control samples after background subtraction.

Tested Articles:
·  Calculate the blank corrected value ODTT= ODTTraw– ODblank.
·  The OD mean per tissue is calculated.
·  The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100.
·  The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues.
·  The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated.
ODTTraw= optical density test article samples.
ODPC= optical density of test article samples after background subtraction.

Data Correction Procedure for MTT Interfering Compounds
True viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt).
ODtvt = optical density of treated viable tissue
ODkt = optical density of killed tissues
ODtkt = optical density of treated killed tissue
ODukt = optical density of untreated killed tissue (NC treated tissue)

Data Correction Procedure for Colored Compounds
True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt – ODvt.
ODtvt = optical density of treated viable tissue incubated in MTT media
ODvt = optical density of viable tissues incubated in media alone.

Proposed Statistical methods
The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated.

- Evaluation of data
The results of the assay was evaluated and compared to negative control.

Table: Irritancy Prediction
In VitroResults In VivoPrediction
Mean tissue viability ≤60% Irritant (I) – Category 1 or 2
Mean tissue viability >60% Non-irritant (NI) – No Category

- Assay quality controls
- Negative Controls (NC)
The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density (OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.
 
- Positive Controls (PC)
Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.
 
- Standard Deviation (SD)
Each test of ocular irritancy potential is predicted from the mean viability determined on 3 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the replicates is <18% for three replicate tissues.

 

Irritation parameter:

other: mean % tissue viability

Run / experiment:

Run 1

Value:

107.7

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 22.1% (for 30 minute exposures with liquids) of negative control in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 17.6 passing the acceptance criteria.

Interpretation of results:

other: not irritating

Conclusions:

The ocular irritation potential of test article Geranyl butyrate (CAS No.106-29-6) was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance Geranyl butyrate was determined to be 107.7%. Thus, substance Geranyl butyrate (CAS No.106-29-6) is considered to be "not irritating" to the human eye.

Executive summary:

The ocular irritation potential of test article Geranyl butyrate (CAS No.106-29-6) was determined according to the OECD 492 test guideline followed for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to liquid test article and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak.  The viability of each tissue was determined by MTT assay. 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 22.1% (for 30 minute exposures) of negative control in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 17.6 passing the acceptance criteria.

The mean % tissue viability of test substance Geranyl butyrate (CAS No.106-29-6) was determined to be 107.7%.

Hence, under the experimental test condition it was concluded that test substance Geranyl butyrate (CAS No.106-29-6) is considered to be not irritating to the human eye and being classified as “Not classified’’ as per CLP Regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation:

In different studies, Geranyl butyrate(CAS No.106-29-6) has been investigated for potential for dermal irritation to a greater or lesser extent. The studies are based on in vitro and in vivo experiments in rabbits along with human data for target chemical as well as for Read across substance Citronellyl Isobutyarte(CAS No.97-89-2).

The dermal irritation potential of test article Geranyl butyrate (CAS No.- 106-29-6) was determined according to the OECD 439 test guideline followed for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article Tissues were exposed to test articls and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 1.3 passing the acceptance criteria.

The Mean % tissue viability compared to negative control (n=3) of the test substance Geranyl butyrate was determined to be 102.5%.

Hence, under the experimental test conditions it was concluded that test substance Geranyl butyrate (CAS No.- 106-29-6) was considered to be not irritating to the skin of humans and being classified as “Not Classified'' as per CLP Regulation.

Various studies were summarized in Food and Cosmetics Toxicology Volume 12, Issues 7–8, December 1974, Pages 889 to evaluate the dermal irritation potential of Geranyl butyrate in vivo experiments in rabbits and humans.

A skin irritation test was conducted on human subjects for test chemical Geranyl butyrate (CAS No: 106-29-6) to determine its irritation potential. During the test, 4% concentration of Geranyl butyrate in petrolatum was applied under occlusion for 48 hours on each subject and then observations were made. None of the subject produced any skin irritation reaction. Hence the test chemical Geranyl butyrate (CAS No: 106-29-6) was considered to be not irritating on skin of humans. A skin irritation test was conducted on rabbits for test chemical Geranyl butyrate (CAS No: 106-29-6) to determine its irritation potential. In this test, Geranyl butyrate applied full strength to intact or abraded rabbit skin for 24 hours under occlusion and then observations were made. No skin reactions were observed in test rabbits. Hence the test chemical Geranyl butyrate (CAS No: 106-29-6) was considered to be not irritating on skin of rabbits.

Skin irritation effects were estimated by three different models i.e., Battery, Lead scope and SciQSAR used within Danish QSAR database for chemical Geranyl butyrate. Based on estimation No severe skin irritation effect were known when test chemical Geranyl butyrate exposed to rabbit skin.

The skin irritation potential for Geranyl butyrate (CAS No: 106-29-6) is estimated using OECD QSAR toolbox version 3.4. The test substance Geranyl butyrate (CAS No: 106-29-6) is estimated to be not irritating to skin of New Zealand White rabbits.

A skin irritation test was conducted (Food and Cosmetics Toxicology Volume 16, Supplement 1, 1978, Pages 693) on human subjects for the similar substance 97-98-2 to assess its irritation potential. During the test, 4% concentration of Citronellyl isobutyrate in petrolatum was applied under occlusion for 48 hours on each subject and then observations were made. There was no irritation seen in any of the subjects. Hence the test chemical Citronellyl isobutyrate (CAS No: 97-98-2) was considered to be not irritating on skin of humans.

Based on the data for the target substance and read across chemical, Geranyl butyrate can be considered as not irritating to skin.        

Eye Irritation:

In different studies, Geranyl butyrate (CAS No.106-29-6) has been investigated for potential for occular irritation to a greater or lesser extent. The studies are based on in vitro and in vivo experiments in rabbits along with human data for target chemical as well as for Read across substance Citronellyl Isobutyarte (CAS No.97-89-2)

The ocular irritation potential of test article Geranyl butyrate (CAS No.106-29-6) was determined according to the OECD 492 test guideline followed for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to liquid test article and controls for ~30 minutes, followed by a ~12 minute post-soak and approximately 2 hour recovery after the post-soak.  The viability of each tissue was determined by MTT assay. 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 22.1% (for 30 minute exposures) of negative control in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 17.6 passing the acceptance criteria.

The mean % tissue viability of test substance Geranyl butyrate (CAS No.106-29-6) was determined to be 107.7%.

Hence, under the experimental test condition it was concluded that test substance Geranyl butyrate (CAS No.106-29-6) is considered to be not irritating to the human eye and being classified as “Not classified’’ as per CLP Regulation.

The ocular irritation potential of Geranyl butyrate was estimated using OECD QSAR toolbox v3.4. Geranyl butyrate was estimated to be not irritating to New Zealand White rabbit eyes.

A primary ocular irritation study was conducted (International Journal of Toxicology; 31(Suppl 1):5-76, 2012) in 6 New Zealand White rabbits to evaluate the irritation potential of the similar substance 7491-02-3 (diisopropyl sebacate). A volume of 0.1 ml diisopropyl sebacate was instilled in to the eyes of each rabbit, which remained unwashed. The other (untreated) eye of each rabbit served as control. The ocular reactions were scored according to Draize method. The average Draize scores were 2.0 at 24 and 48 hours, 0.3 at 72 hours and 0.0 at 4 days. Based on the scores after 4 days, diisopropyl sebacate can be considered as not irritating to New Zealand White rabbit eyes.

A rabbit eye irritation study was conducted (Monographs on Fragrance Raw Materials, A Collection of Monographs Originally Appearing in Food and Cosmetics Toxicology, 1979, Pages 404) to assess the irritation potential of the similar substance 105-86-2 (Geranyl formate). Geranyl formate was instilled into rabbit eyes (number not specified) and the effects were observed for 72 hours. Slight irritation was observed in rabbits which disappeared after 72 hours. Hence Geranyl formate can be considered as not irritating to rabbit eyes.

Based on the available information for the target as well as its read across chemicals , Geranyl butyrate can be considered as not irritating to eyes.  

Justification for selection of skin irritation / corrosion endpoint:

data is from study reportl having Klimisch rating 1

Justification for selection of eye irritation endpoint:

data is from study reportl having Klimisch rating 1

Justification for classification or non-classification

Available studies for the target chemical indicate that Geranyl butyrate was not irritating to eyes and skin.Hence Geranyl butyrate can be evaluated as “Non Irritant” to eyes and skin.