Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Test System
The EpiAirway™ Model provided by MatTek Corporation (Ashland, MA, USA) was used in this study. The EpiAirway™ tissue is a culture of normal human bronchial epithelial cells (NHBE) grown at air-liquid interface (ALI). The EpiAirway™ tissues are pseudo-stratified containing ciliated epithelial cells, basal cells, and mucus producing goblet cells. The epithelial lining of the human respiratory tract is pseudo-stratified containing ciliated epithelial cells and mucus producing cells goblet cells from the upper respiratory section down through to the bronchus (Bérubé, et al. 2009).
Experimental Design and Methodology
The experimental design of this study consisted of a determination of the direct MTT reduction potential and the pH of the test article followed by a definitive assay to determine the relative tissue viability. Up to four concentrations of the test article were treated onto the EpiAirway™ tissues for 3 hours. The toxicity of the test article was determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and, to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control, and test article -treated cultures (Berridge et al., 1996). Data was presented in the form of relative survival (MTT conversion relative to the negative/solvent control). An EC75 (the concentration of the test article required to reduce tissue viability to 75% of the negative/solvent control) was determined for the test article.
GLP compliance:
yes (incl. certificate)
Test type:
other: NAD(P)H-dependent microsomal enzyme reduction of MTT for 4 concentrations of test article
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Substance %
Isopropyl Jojobate 30 – 40
Jojoba Alcohols 30 – 40
Jojoba Esters 30 – 40

Test animals

Species:
other: normal human bronchial epithelial cells

Administration / exposure

Route of administration:
other: topical administration
Type of inhalation exposure:
other: topical administration
Vehicle:
other: corn oil
Control animals:
yes
Remarks:
egative control (sterile deionized water) and positive control (14.7 mg/mL formaldehyde)
Details on study design:
Test System
The EpiAirway™ Model provided by MatTek Corporation (Ashland, MA, USA) was used in this study. The EpiAirway™ tissue is a culture of normal human bronchial epithelial cells (NHBE) grown at air-liquid interface (ALI). The EpiAirway™ tissues are pseudo-stratified containing ciliated epithelial cells, basal cells, and mucus producing goblet cells. The epithelial lining of the human respiratory tract is pseudo-stratified containing ciliated epithelial cells and mucus producing cells goblet cells from the upper respiratory section down through to the bronchus (Bérubé, et al. 2009).

Experimental Design and Methodology
The experimental design of this study consisted of a determination of the direct MTT reduction potential and the pH of the test article followed by a definitive assay to determine the relative tissue viability. Up to four concentrations of the test article were treated onto the EpiAirway™ tissues for 3 hours. The toxicity of the test article was determined by the NAD(P)H-dependent microsomal enzyme reduction of MTT (and, to a lesser extent, by the succinate dehydrogenase reduction of MTT) in control, and test article -treated cultures (Berridge et al., 1996). Data was presented in the form of relative survival (MTT conversion relative to the negative/solvent control). An EC75 (the concentration of the test article required to reduce tissue viability to 75% of the negative/solvent control) was determined for the test article.

Test Article Preparation
Each test article was administered to the test system prepared in sterile, corn oil. Each test article dilution was prepared by weighing the test article in a pre-labeled conical tube. Sterile, corn oil was added until the dilutions were achieved and the tube was vortexed for approximately 1 minute prior to application. For the remainder of this report, each test article dilution is referred to as the test article.

Route of Administration
The test article and assay controls were applied topically to the tissue. One hundred microliters (100 μL) of each test article concentration and control, respectively, were applied to each tissue. Three tissues were used for each test article concentration or control. After dosing, an insert cap was applied to each cell culture insert.

pH Determination
The pH value of the highest dosing dilution was determined using pH paper (EMD Millipore Corporation). For both the preliminary and definitive assays, the highest dose of each test article were added to pH paper with a 0-14 pH range in 1.0 pH increments to approximate a narrow pH range. Next, the test articles were added to pH paper with a narrower range of 0-6 pH or 7-10 pH with 0.5 pH unit increments, to obtain a more accurate pH value. The pH values obtained from the preliminary assay are presented in Table 1. The pH values obtained from the definitive assay are presented in Table 2.

Controls
The definitive assay included a negative (sterile deionized water) and solvent control (Corn Oil). A reference control, formaldehyde, was also tested concurrently.
Assessment of Direct Test Article Reduction of MTT
Each test article was added to a 1.0 mg/mL MTT solution in warm Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2 mM L-glutamine (MTT Addition Medium) to assess its ability to directly reduce MTT. Approximately 100 μL of each test article was added to 1 mL of the MTT solution and the mixtures were incubated in the dark at standard culture conditions for one hour. A negative control, 100 μL of sterile, deionized water, was tested concurrently. If the MTT solution color turned blue/purple, the test article was presumed to have reduced the MTT. The test articles were observed to reduce MTT directly in the absence of viable cells. A killed control experiment was performed concurrently in the screening and definitive assays to determine the extent of the direct MTT reduction (if any) by the test articles alone.

Receipt of the EpiAirway™ Human Cell Construct Model
Upon receipt of the EpiAirway™ Human Cell Construct Kit, the solutions were stored as indicated by the manufacturer. The EpiAirway™ human cell constructs were stored at 2-8ºC until used.
The day before dosing, an appropriate volume of EpiAirway™ assay medium (AM) was warmed at 37±1ºC. One milliliter (1 mL) of AM was aliquoted into the wells of 6-well plates. Each EpiAirway™ tissue was inspected for air bubbles between the agarose gel and cell culture insert prior to opening the sealed package. Cultures with air bubbles greater than 50% of the cell culture insert area were not used. The 24-well shipping containers were removed from the plastic bag and the surface disinfected with 70% ethanol. An appropriate number of EpiAirway™ tissues were transferred aseptically from the 24-well shipping containers into the 6-well plates.
The EpiAirway™ tissues were incubated at 37±1ºC in a humidified atmosphere of 5±1% CO2 in air (standard culture conditions) overnight (at least 16 hours), to acclimate the tissue. Upon opening the bag, any unused tissues remaining on the shipping agar were briefly gassed with an atmosphere of 5% CO2/95% air, and the bag was sealed and stored at 2-8ºC for subsequent use.

Preliminary Assay
The test articles: Jojoba Esters and, Isopropyl Jojobate and Jojoba Alcohol and Jojoba Esters, and Hydrolyzed Jojoba Esters, negative control (sterile deionized water) and positive control (14.7 mg/mL formaldehyde) were tested in the preliminary assay (16 May 2018).
At least 16 hours after initiating the tissues, the medium was removed from under the tissue and replaced with 1 mL of fresh pre-warmed AM. Immediately after refeeding, the tissues’ apical surface was rinsed twice with 0.4 mL of sterile Ca++Mg++Free-DPBS (CMF-DPBS) (warmed at 37°C) to remove a mucus layer that may be coating the apical surface of the tissues. The CMF-DPBS was added slowly to the apical side of the tissue (directed at the side of the cell culture insert, i.e. not directly onto the tissue surface) and then gently aspirated. The tissues were inverted and gently blotted on sterile absorbent to remove excess moisture.
Three concentrations (500, 250, and 50 mg/mL) were selected for each test article. One hundred μL of each test article were applied in a single EpiAirway™ tissue per concentration for 3 hours. A single tissue was treated with 100 μL of the negative and positive control for 3 hours. The tissues were incubated at standard culture conditions before being transferred to MTT.

MTT Assay
A 10X stock of MTT prepared in PBS (filtered at time of batch preparation) was thawed and diluted in warm MTT Addition Medium to produce the 1.0 mg/mL solution no more than two hours before use. Three hundred μL of the MTT solution was added to each designated well of a pre-labelled 24-well plate. After 3±0.1 hours, the insert caps were removed from the cell culture insert, and each EpiAirway™ tissue was rinsed with CMF-DPBS. Each tissue was rinsed three times with 0.4 mL (per rinse) of CMF-DPBS. The CMF-DPBS was gently pipetted into the well and then aspirated off. Care was exercised to avoid touching the surface of the tissue. The EpiAirway™ tissues were blotted on absorbent paper and then transferred to the appropriate wells containing MTT. The 24-well plates were incubated at standard conditions for 3 ± 0.1 hours. After approximately three hours, the EpiAirway™ tissues were blotted on absorbent paper, cleared of excess liquid, and transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well. Then the plates were shaken for at least 2 hours at room temperature. At the end of the extraction period, the liquid within the culture inserts was decanted into the well from which the insert was taken. The extracted solution was mixed and 200 μL transferred to the appropriate wells of 96-well plate. Two hundred μL of isopropanol was added to the wells designated as blanks. The absorbance at 550 nm (OD550) of each well was measured with a Molecular Devices Vmax plate reader.

Killed Controls (KC)
To evaluate whether residual test article was binding to the tissue and leading to a false MTT reduction signal, a functional check (using freeze-killed control tissue) was performed. Freeze killed tissues were received already prepared from MatTek, and were stored in the freezer until use. A single KC tissue was treated with each test article at 500 mg/mL for 3 hours during the preliminary assay.
Little or no MTT reduction was observed in the test article-treated KC tissues (net OD<0.102. The results of the preliminary assay necessitated the use of higher test article concentrations for the definitive assay.
For the definitive assay, a single KC tissue was treated with each test article at the neat concentration. A single KC was treated with the solvent (corn oil) and negative control (sterile deionized water) for 3 hours. The raw OD value of the solvent KC was 0.060, which was identical to the raw OD value of the negative control. None of the OD values for the test articles were considered significant.

Definitive Assay
The definitive assay was performed (17 May 2018) for the test articles: Jojoba Esters, Isopropyl Jojobate and Jojoba Alcohol and Jojoba Esters, and Hydrolyzed Jojoba Esters, a solvent control (corn oil), negative control (sterile deionized water), and positive control (14.7 mg/mL formaldehyde) were also tested.
At least 16 hours after initiating the tissues, the medium was removed from under the tissue and replaced with 1 mL of fresh, pre-warmed AM. Immediately after refeeding, the tissues’ apical surface was rinsed twice with 0.4 mL of sterile Ca++Mg++Free-DPBS (CMF-DPBS) (warmed at 37°C) to remove a mucus layer that may be coating the apical surface of the tissues. The CMF-DPBS was added slowly to the apical side of the tissue (directed at the side of the cell culture insert, i.e. not directly onto the tissue surface) and then gently aspirated. The tissues were inverted and gently blotted on sterile absorbent to remove excess moisture.
Four concentrations (neat, 850, 650, and 450 mg/mL) were selected for each test article. All treatment groups were dosed topically in triplicate tissues with 100 μL of the test article or controls. After dosing, an insert cap was applied onto each cell culture insert. The tissues were incubated for 3±0.1 hours at standard culture conditions. At the conclusion of the incubation period, tissues were transferred to MTT and processed as previously described.

Data Analysis
All calculations were performed using an Excel spreadsheet. The mean OD550 value of the blank wells was calculated. The corrected mean OD550 value of the negative control, solvent control, reference control and test articles were determined by subtracting the mean OD550 value of the blank wells from their mean OD550 values. Calculations were performed according to the following equation:
(see report)
The usage of killed controls required additional calculations to correct for the amount of MTT reduced directly by test article residues. The raw OD550 for the negative control was subtracted from the raw OD550 values for each of the test article-treated killed controls, to determine the net OD550 values for the test article treated killed controls. Calculations were performed according to the following equation:
(see report)
The net OD550 values represent the amount of reduced MTT due to direct reduction by test article residues. If the net OD550 value is greater than 0.150, the net amount of MTT reduction is subtracted from the corrected OD550 values of the viable treated tissues to obtain a corrected OD550 value. The final corrected OD550 values were then used to determine the % of negative control viabilities. Calculations were performed according to the following equation:
(see report)
Finally, the following % of Control calculation was made:
(see report)
The individual % of Control values were then averaged to calculate the mean % of Control. Test article and reference control viability calculations were performed by comparing the corrected OD550 values of the test article or reference control to the negative control.
Concentration response curves were plotted with the % of control on the ordinate and the test article concentration on the abscissa. The EC75 value was interpolated from each plot. To determine the EC75 value, two consecutive points were selected where one concentration results in a relative survival greater than 70% and one concentration results in less than 75% viability. The two select exposures were used to determine the slope and the y-intercept for the equation y = m(x) + b. To determine the EC75 value, the equation was solved for y = 75. If all of the concentrations show greater than 75% survival, the EC75 value was presented as greater than the maximum concentration. If all of the concentrations show less than 75% survival, the EC75 value was extrapolated assuming a 0% concentration as 100% viability.
Statistics:
Criteria for a Valid Test
The assay was considered acceptable if the positive control (14.7 mg/mL formaldehyde) fell within the acceptance criteria as defined by the tissue manufacturer. The criteria are as follows: OD550 of <75% of negative control.
Evaluation of Test Results
The prediction models presented in Jackson et al. (2016) were used to evaluate the acute toxicity potential. According to the prediction models, an EC75 value less than 150 mg/mL or 200 mg/mL will be consistent with an EPA irritant (Category I-III) or GHS irritant (Category 1-3), respectively. EC75 values greater than 150 mg/mL or 200 mg/mL will be consistent with an EPA Category IV or GHS Category 4-5, respectively.
EC75 results should be carefully evaluated based on the toxicity profile and doses tested.

Results and discussion

Preliminary study:
and Hydrolyzed Jojoba Esters, negative control (sterile deionized water) and positive control (14.7 mg/mL formaldehyde) were tested in the preliminary assay (16 May 2018).
At least 16 hours after initiating the tissues, the medium was removed from under the tissue and replaced with 1 mL of fresh pre-warmed AM. Immediately after refeeding, the tissues’ apical surface was rinsed twice with 0.4 mL of sterile Ca++Mg++Free-DPBS (CMF-DPBS) (warmed at 37°C) to remove a mucus layer that may be coating the apical surface of the tissues. The CMF-DPBS was added slowly to the apical side of the tissue (directed at the side of the cell culture insert, i.e. not directly onto the tissue surface) and then gently aspirated. The tissues were inverted and gently blotted on sterile absorbent to remove excess moisture.
Three concentrations (500, 250, and 50 mg/mL) were selected for each test article. One hundred μL of each test article were applied in a single EpiAirway™ tissue per concentration for 3 hours. A single tissue was treated with 100 μL of the negative and positive control for 3 hours. The tissues were incubated at standard culture conditions before being transferred to MTT.
Effect levels
Key result
Sex:
not specified
Dose descriptor:
other: EC75
Effect level:
> 1 000 other: mg/mL
Based on:
test mat. (total fraction)
Exp. duration:
3 h
Remarks on result:
not determinable due to absence of adverse toxic effects
Other findings:
Summary
The acute inhalation toxicity potential of the test articles were evaluated in EpiAirway™ tissues using the methods and prediction model presented by MatTek.
All tested concentrations of the test articles: Jojoba Esters, Isopropyl Jojobate and Jojoba Alcohol and Jojoba Esters, and Hydrolyzed Jojoba Esters resulted in relative viabilities of ~100%, and therefore the EC75 was presented as greater than the highest tested dose.
According to the prediction models, an EC75 value greater than 150 mg/mL or 200 mg/mL would be consistent with an EPA Category IV or GHS Category 4-5, respectively. The test articles: Jojoba Esters, Isopropyl Jojobate and Jojoba Alcohol and Jojoba Esters, and Hydrolyzed Jojoba Esters would be predicted as EPA Category IV/GHS Category 4-5.
Deviation
Protocol Amendment II states that during the preliminary assay a single killed control tissue would be treated with the negative or solvent control in parallel with the killed control tissues that received test article. The killed control tissue that would have received the negative or solvent control was not tested. However, the Optical Density (O.D.) values for the killed controls treated with the test article run during the preliminary assay were all below the level of significance, suggesting the absence of the negative or solvent killed control tissue did not affect the preliminary study. Negative and solvent control killed control tissues were run during the definitive assay, the O.D. values were also below the level of significance.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The acute inhalation toxicity potential of the test articles were evaluated in EpiAirway™ tissues using the methods and prediction model presented by MatTek.
All tested concentrations of the test articles: Jojoba Esters, Isopropyl Jojobate and Jojoba Alcohol and Jojoba Esters, and Hydrolyzed Jojoba Esters resulted in relative viabilities of ~100%, and therefore the EC75 was presented as greater than the highest tested dose.
According to the prediction models, an EC75 value greater than 150 mg/mL or 200 mg/mL would be consistent with an EPA Category IV or GHS Category 4-5, respectively.

The test articles: Jojoba Esters, Isopropyl Jojobate and Jojoba Alcohol and Jojoba Esters, and Hydrolyzed Jojoba Esters would be predicted as EPA Category IV/GHS Category 4-5.
As the test did not highlight any sign of toxicity, the registrant choses not to classify the substance awaiting for further testing.
Executive summary:

The acute inhalation toxicity potential of the test articles were evaluated in EpiAirway™ tissues using the methods and prediction model presented by MatTek.

All tested concentrations of the test articles: Jojoba Esters, Isopropyl Jojobate and Jojoba Alcohol and Jojoba Esters, and Hydrolyzed Jojoba Esters resulted in relative viabilities of ~100%, and therefore the EC75 was presented as greater than the highest tested dose.

According to the prediction models, an EC75 value greater than 150 mg/mL or 200 mg/mL would be consistent with an EPA Category IV or GHS Category 4-5, respectively.

As the test did not highlight any sign of toxicity, the registrant choses not to classify the substance awaiting for further testing.

The test articles: Jojoba Esters, Isopropyl Jojobate and Jojoba Alcohol and Jojoba Esters, and Hydrolyzed Jojoba Esters would be predicted as EPA Category IV/GHS Category 4-5.