The Antimicrobial and Toxicity Influence of Six Carrier Oils on Essential Oil Compounds

Essential oil compounds have been identified as alternative antimicrobials; however, their use is limited due to their toxicity on human lymphocytes, skin, and reproduction. Carrier oils can reduce the toxicity of essential oils, which raises the question as to whether such activity would extend to the essential oil compounds. Thus, this study aimed to investigate the antimicrobial and toxicity activity of essential oil compounds in combination with carrier oils. The antimicrobial properties of the essential oil compounds, alone and in combination with carrier oils, were determined using the broth microdilution assay. The toxicity was determined using the brine shrimp lethality assay. Antimicrobial synergy (ΣFIC ≤ 0.50) occurred in 3% of the samples when tested against the ESKAPE pathogens. The compound thymoquinone in combination with the carrier oil Prunus armeniaca demonstrated broad-spectrum synergistic activity and a selectivity index above four, highlighting this combination as the most favorable. The carrier oils reduced the toxicity of several compounds, with Calendula officinalis and P. armeniaca carrier oils being responsible for the majority of the reduced toxicity observed. This study provides insight into the interactions that may occur when adding a carrier oil to essential oil compounds.


Introduction
Antimicrobial resistance is responsible for a large number of morbidities and mortalities worldwide [1,2]. The World Health Organization published a list of priority microorganisms known as the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli) which requires urgent attention with respect to antimicrobial resistance [3]. Essential oils have been identified as alternative antimicrobial options due to their observed antimicrobial properties against a wide range of pathogens, especially against Gram-positive micro-organisms [4][5][6][7][8][9][10].

Combinations
The MIC values of the 21 compounds in combination with six carrier oils against seven pathogens (882 combinations in total) were determined, and the results are shown in Tables 3-9. In summary, most of the combinations resulted in indifference (56%), followed by 37% additive interactions. There was a total of 3% synergistic (23 combinations) and 4% antagonistic interactions noted.
The combined use of A. vera, C. officinalis, and H. perforatum with α-terpinene resulted in the most synergy against E. faecium (ΣFIC value of 0.41) ( Table 3). The compound α-terpinene was present in 75% of the synergistic interactions against E. faecium. The combined use of thymoquinone with H. perforatum resulted in the most antagonistic interaction against E. faecium (ΣFIC value of 12.01), and the compound thymoquinone was present in 83% of the antagonistic interactions against E. faecium.
Against S. aureus, the combination of thymoquinone with H. perforatum resulted in the most synergistic interaction (ΣFIC value of 0.13), and the compound thymoquinone was present in most of the synergistic interactions (46%), displaying synergy when combined with all six carrier oils tested ( Table 4). The combination of santalol with A. vera displayed the highest antagonistic interaction against S. aureus (ΣFIC value of 8.75).
None of the compound: carrier oil combinations displayed synergistic interactions against K. pneumoniae, and one combination (santalol with P. americana) displayed antagonism (ΣFIC value of 6.00) ( Table 5).       None of the compound: carrier oil combinations resulted in synergy against A. baumannii (Table 6). Antagonism was apparent for combinations of α-terpinene with P. americana, and (+)-α-pinene with P. armeniaca (ΣFIC values of 6.67). The carrier oils P. americana and P. armeniaca were present most often in antagonistic interactions (40%).
None of the compound: carrier oil interactions displayed synergistic interactions against P. aeruginosa ( Table 7). The combination of p-cymene with A. vera was the only combination which resulted in antagonism (ΣFIC value of 7.24).
The combination demonstrating the most synergy against E. coli was thymoquinone and A. vera (ΣFIC value of 0.09) ( Table 8). Thymoquinone was present in all of the synergistic interactions observed. The combinations which showed the most antagonism was p-cymene combined with S. chinensis and linalool combined with S. chinensis (ΣFIC values of 4.95). The compound santalol (29%) and carrier oil S. chinensis (57%) were present most often in antagonistic interactions.
The combination of p-cymene with H. perforatum and β-caryophyllene with P. armeniaca resulted in synergistic interactions against C. albicans (ΣFIC values of 0.50) ( Table 9). None of the combinations resulted in antagonism.
In summary (Figure 1), the compound thymoquinone and the carrier oil P. armeniaca were present in the majority of the synergistic combinations. The carrier oil H. perforatum and the compound santalol were present most frequently in the antagonistic combinations.
Of the four compounds (carvacrol, cinnamaldehyde, isoeugenol, and thymol) which showed broad-spectrum, noteworthy antimicrobial activity against all reference strains tested, only thymol produced some synergistic antimicrobial activity when combined with carrier oils. This suggests that noteworthy antimicrobial activity of a compound by itself does not necessarily correlate to synergy when combined with carrier oils. In fact, the compounds cinnamaldehyde, citral, santalol, and thymoquinone, which showed noteworthy MIC values when tested alone, were present in several antagonistic combinations when combined with carrier oils. Thymoquinone and santalol in particular were present repeat-edly in antagonistic combinations against more than one reference strain. The influence of the carrier oil on the antimicrobial activity of a compound differed according to the reference strain tested. When observing the interactive profiles of the compound: carrier oil combinations against the various reference strains tested, it was noted that the combinations tested against the Gram-positive bacteria displayed the highest synergy (6%) as well as the highest antagonism (6%). The combinations tested against the Gram-negative bacteria displayed the least synergy (1%), and combinations tested against the yeast reference strain displayed the second highest synergy (2%) and the least antagonism (0%). Thymoquinone was the compound most commonly observed in the synergistic antimicrobial interactions.
In another study [31] where essential oils were combined with carrier oils against skin pathogens, most of the synergistic interactions also occurred against the Gram-positive bacteria. The enhanced susceptibility of the Gram-positive bacteria may be due to the susceptibility of the outer membrane where the structure is less complex than that of the Gram-negative micro-organisms. The structure consists of a membrane weaker than that of the Gram-negative bacteria and consists only of a thick peptidoglycan wall which is not adequate to prevent the entry of antimicrobial compounds [50,51].
The antimicrobial enhancing properties of the carrier oils present in the synergistic combinations could be attributed to their free fatty acids [31]. Free fatty acids, such as oleic and linoleic acid, have been reported to show antimicrobial activity against the Grampositive micro-organism S. aureus when tested at high concentrations [52]. The antimicrobial activity of free fatty acids could be attributed to their ability to cause cell lysis, disruption to nutrient uptake, inhibition of enzyme activity, and formation of auto-oxidation products, as well as their ability to alter pH levels, thus causing disturbance to the bacterial membrane [53,54].
In a previous study [31], combinations of essential oils with the carrier oils A. vera, H. perforatum, P. americana, P. armeniaca and S. chinensis resulted in synergy against C. albicans. In this study, combinations of two compounds with H. perforatum and P. armeniaca resulted in synergy. The two compounds which showed synergy against C. albicans were present as major compounds in the essential oils Kunzea ericoides A.Rich. Joy Tomps. (kanuka) and Lavandula angustifolia Mill. (lavender) which also resulted in synergy with the carrier oils [31]. This demonstrates that there were instances where the synergy observed for a single compound: carrier oil combination correlated with the synergistic interaction observed by the neat essential oil: carrier oil combination. As an example, K. When observing the interactive profiles of the compound: carrier oil combinations against the various reference strains tested, it was noted that the combinations tested against the Gram-positive bacteria displayed the highest synergy (6%) as well as the highest antagonism (6%). The combinations tested against the Gram-negative bacteria displayed the least synergy (1%), and combinations tested against the yeast reference strain displayed the second highest synergy (2%) and the least antagonism (0%). Thymoquinone was the compound most commonly observed in the synergistic antimicrobial interactions.
In another study [31] where essential oils were combined with carrier oils against skin pathogens, most of the synergistic interactions also occurred against the Gram-positive bacteria. The enhanced susceptibility of the Gram-positive bacteria may be due to the susceptibility of the outer membrane where the structure is less complex than that of the Gram-negative micro-organisms. The structure consists of a membrane weaker than that of the Gram-negative bacteria and consists only of a thick peptidoglycan wall which is not adequate to prevent the entry of antimicrobial compounds [50,51].
The antimicrobial enhancing properties of the carrier oils present in the synergistic combinations could be attributed to their free fatty acids [31]. Free fatty acids, such as oleic and linoleic acid, have been reported to show antimicrobial activity against the Gram-positive micro-organism S. aureus when tested at high concentrations [52]. The antimicrobial activity of free fatty acids could be attributed to their ability to cause cell lysis, disruption to nutrient uptake, inhibition of enzyme activity, and formation of autooxidation products, as well as their ability to alter pH levels, thus causing disturbance to the bacterial membrane [53,54].
In a previous study [31], combinations of essential oils with the carrier oils A. vera, H. perforatum, P. americana, P. armeniaca and S. chinensis resulted in synergy against C. albicans. In this study, combinations of two compounds with H. perforatum and P. armeniaca resulted in synergy. The two compounds which showed synergy against C. albicans were present as major compounds in the essential oils Kunzea ericoides A.Rich. Joy Tomps. (kanuka) and Lavandula angustifolia Mill. (lavender) which also resulted in synergy with the carrier oils [31]. This demonstrates that there were instances where the synergy observed for a single compound: carrier oil combination correlated with the synergistic interaction observed by the neat essential oil: carrier oil combination. As an example, K. ericoides essential oil, containing p-cymene (11.9%) as a major compound, resulted in synergy when combined with A. vera, and p-cymene combined with A. vera resulted in synergy in this study. This was also observed with L. angustifolia essential oil, containing linalyl acetate (35.6%), which resulted in synergy when combined with C. officinalis; and in this study, the combination of linalyl acetate with C. officinalis also resulted in synergy.
Very few synergistic interactions were observed against the Gram-negative bacteria previously [31], and in this study. The combinations which did result in synergy against Gramnegative bacteria were thymoquinone combined with A. vera, P. americana, P. armeniaca, and S. chinensis. Antagonism was seen most frequently against the Gram-negative bacteria by the compound santalol and the carrier oils P. americana and S. chinensis.

Toxicity Analysis
All six carrier oils tested were non-toxic at both 24 and 48 h ( Table 10). The least toxic of the carrier oils was P. americana. These results are congruent with a previous carrier oil study [31]. 1 Bold values represent biological non-toxicity; shaded area shows carrier oils and non-shaded area shows compounds. 2 Although acetone is known as a toxic agent to brine shrimp, it was the only solvent that allowed dilution of several insoluble compounds; thus, diluted acetone was used and included as a negative control.
At 24 h, 24% of the compounds showed non-toxic results and 19% of the compounds showed non-toxic results at 48 h. At both 24 and 48 h, the compounds β-caryophyllene, p-cymene, linalyl acetate, and γ-terpinene were non-toxic, and R (+)-limonene was non-toxic only at 24 h. The compounds p-cymene (at 24 h), linalyl acetate, and γ-limonene showed non-toxicity in previous studies [55][56][57]. The other compounds (76% and 81%) showed toxicity to the brine shrimp either at 24 h or, at both 24 and 48 h, showing the highly toxic nature of the compounds by themselves, even when diluted to a concentration of 1.00 mg/mL.

Combinations
After combining the 21 compounds with all six carrier oils (Tables 11-16), it was found that in several instances the toxicity of the compounds was reduced. At 24 h, the combinations containing C. officinalis, H. perforatum, and P. armeniaca resulted in the most reduction in compound toxicity, and at 48 h, H. perforatum resulted in the most reduction in compound toxicity. The carrier oil H. perforatum would therefore be a suitable option to be combined with compounds tested for the purpose of reducing toxicity. Table 11. Mean toxicity (% mortality), standard deviation (SD), ΣFPM (fractional percentage mortality index), and interpretation of essential oil compound: Aloe vera combinations (n = 3). The combination of p-cymene with A. vera resulted in the most favorable synergistic interaction at 24 h (ΣFIC value of 0.28) (Table 11), and α-terpinene when combined with A. vera resulted in the only synergistic interaction observed at 48 h. The highest antagonistic ΣFIC values at 24 and 48 h resulted from the combination of γ-terpinene with A. vera (ΣFIC values of 49.32 and 18.01 respectively).

Essential Oil Compound
At 24 h, when R (+)-limonene and γ-terpinene were combined with C. officinalis, a complete reduction in toxicity was observed (Table 12)   Incr/decr toxicity (increase/decrease toxicity)-the increase or decrease in toxicity of the compound from when tested alone to when combined with the carrier oil. 2 Int (interpretation)-the interpretation of ΣFIC values, whether antagonistic (Ant) (italics), synergistic (Syn) (bold and italics), additive (Add), or indifferent (Ind). 3 Bold values represent biological non-toxicity. 4 Value could not be calculated due to the carrier oil's toxicity being 0.00%. Table 15. Mean toxicity (% mortality), standard deviation (SD), ΣFPM, and interpretation of essential oil compound: Prunus armeniaca combinations (n = 3). Incr/decr toxicity (increase/decrease toxicity)-the increase or decrease in toxicity of the compound from when tested alone to when combined with the carrier oil. 2 Int (interpretation)-the interpretation of ΣFIC values, whether antagonistic (Ant) (italics), synergistic (Syn) (bold and italics), additive (Add), or indifferent (Ind). 3 Bold values represent biological non-toxicity.

Essential Oil Compound
The combined use of santalol and H. perforatum was the only synergistic interaction at 24 h, and at 48 h γ-terpinene and H. perforatum resulted in the most synergistic interaction (ΣFIC value of 0.42) (Table 13). Several compound: carrier oil combinations resulted in antagonistic ΣFIC values at 24 and 48 h.
The toxicity of P. americana alone at 24 h was 0.00% and so ΣFIC values at 24 h could not be calculated; however, it could be noted that the toxicity of the compounds isoeugenol, linalool, santalol, α-terpinene, and (+)-terpinen-4-ol reduced from toxic levels to non-toxic levels when combined with P. americana at 24 h. None of the compound: carrier oil combinations displayed synergy at 48 h (Table 14). The combination of γ-terpinene and H. perforatum was the most antagonistic (ΣFIC value of 54.52).
At 24 h, the combination of santalol and P. armeniaca resulted in a complete decrease in toxicity, followed by R (+)-limonene and P. armeniaca which resulted in the second most synergistic interaction with an ΣFIC value of 0.20. At 48 h, santalol or R (+)-limonene combined with P. armeniaca resulted in the most synergistic interactions (ΣFIC values of 0.19). At 24 and 48 h, several combinations were antagonistic ( Table 15).
All of the compounds with S. chinensis resulted in antagonism at 24 h (Table 16). Less antagonism was observed at 48 h, where the most antagonistic ΣFIC value resulted from linalyl acetate combined with S. chinensis (ΣFIC value of 4.89). Table 17 provides a summary of the toxicity percentage of the interactions of each carrier oil in combination with the essential oil compounds. Synergy indicates that the carrier oil was able to quench the toxicity of the essential oil compounds, rendering it non-toxic. The carrier oil P. armeniaca resulted in the most synergy in its respective combinations with the compounds at 48 h. A constituent of P. armeniaca, vitamin E [31], may be the contributing factor to the carrier oil's favorable toxicity quenching abilities as it was previously reported that vitamin E was able to reduce the toxic effect of the medicine digoxin in rabbits [32] and acute mercury toxicity in rats [70]. At 24 h, the carrier oil S. chinensis resulted in the most antagonism within its combinations, and C. officinalis (responsible for majority of the synergistic interactions) showed the least antagonism. Therefore, at 24 h, C. officinalis would be the most favorable carrier oil choice to be combined with the compounds used in this study to reduce their toxicity. At 48 h, the carrier oil P. americana was responsible for the majority of the antagonistic interactions, and S. chinensis showed the least.
The compounds that most commonly quenched toxicity and therefore resulted in synergistic interactions when combined with the carrier oils at both 24 and 48 h were α-terpinene; linalyl acetate; γ-terpinene; R (+)-limonene; and santalol. The compound R (+)-limonene quenched toxicity the most.
To the best of our knowledge, to date there have been no previous studies conducted on the toxicity of the combined use of essential oil compounds with carrier oils; however, there has been a study on the combined use of essential oils with the same carrier oils as carried out in this study [31]. The synergy was consistent for several of the compounds and essential oils across the two studies. This could be observed for the synergistic combination of p-cymene with A. vera. At 24 h, the essential oils K. ericoides and Melaleuca alternifolia Cheel (tea tree), containing the compound p-cymene (11.9% and 9.6%, respectively), showed synergy when combined with A. vera [31]. This could suggest a correlation between the synergistic activity seen with the essential oil: carrier oil combinations and the synergistic activity seen with the essential oil compound: carrier oil combinations.
The previous study also found the carrier oils A. vera and S. chinensis to reduce the toxicity of the essential oils at 24 h and A. vera and P. armeniaca to cause the most reduction in toxicity at 48 h. Aloe vera was present in most synergistic essential oil-carrier oil combinations over 24 and 48 h [31]. Some differences in the results between this study and the previous one suggests that the toxicity patterns shown by the combined use of carrier oils and essential oils cannot always be generalized to predict which carrier oil would be most advantageous in decreasing the toxicity of the compounds. The essential oil L. angustifolia, containing linalyl acetate (35.6%), linalool (32.8%), and β-caryophyllene (10.2%) as its major compounds, resulted in synergy when combined with C. officinalis [31], and in this study, linalyl acetate resulted in synergy with C. officinalis whereas β-caryophyllene and linalool did not. This observed difference may also be due to the mixture of compounds in the neat essential oil reacting differently when compared to examining combinations with single compounds.

Selectivity Index
The selectivity index for all the combinations which showed antimicrobial synergy was calculated (Table 18). Various interpretations exist, however, this study considers a selectivity index of >4 as being acceptable, when the antimicrobial benefit is not lost due to the toxicity [71]. A selectivity index below four indicates that the toxicity of the compound: carrier oil combination is too high and the antimicrobial activity is most likely attributed to the toxicity of the sample and not the interaction [71]. Of the 23 synergistic combinations, 10 at 24 h and 9 at 48 h had SI values of >4, with thymoquinone being the main compound present in these combinations.

Culture Preparation
The micro-organisms selected for this study included the ESKAPE pathogens and one yeast pathogen. Selection was based on their importance in contributing towards antimicrobial resistance [2,77]. The investigated bacteria included Enterococcus faecium (ATCC 27270), Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 13883), Acinetobacter baumannii (ATCC 17606), Pseudomonas aeruginosa (ATCC 27858), and Escherichia coli (ATCC 8739). The pathogen reference strain Candida albicans (ATCC 10231) was selected as a yeast representative. The micro-organisms were cultured in Tryptone Soya broth (TSB) (Oxoid), and Tryptone Soya agar (TSA) and were incubated at 37 • C for 24 h (bacteria) and at 37 • C for 48 h (yeast). The purity of the micro-organisms was confirmed by streaking each culture onto an agar plate and ensuring growth of single colonies, as well as checking colony morphology with visual standards within the microbiology laboratory.

Sample Preparation
For the broth microdilution assay, the samples were diluted to a concentration of 32.00 mg/mL in acetone. For the brine shrimp lethality assay, all selected samples were prepared in 2% dimethyl sulfoxide (DMSO) or 20-50% acetone at a concentration of 2.00 mg/mL depending on solubility.

Antimicrobial Analysis
The broth microdilution method using a 96-well microtiter plate, as described in a previous study [5], was used to quantify the inhibitory activity of the compounds and carrier oils. Preparation of the microtiter plates involved the aseptic addition of 100.00 µL of TSB into each of the wells of the microtiter plate. The samples were then added, at a volume of 100.00 µL, to the first row of the plate. When testing the combinations, a modification was made where 50.00 µL of the compound and 50.00 µL of the carrier oil were placed in the first row of wells (to make up 100 µL of sample) of the plate. A volume of 100.00 µL of a positive, negative, and culture control were included for each strain studied. The positive control (0.01 mg/mL ciprofloxacin for bacteria or 0.1 mg/mL nystatin for yeast) was used to ensure microbial susceptibility. The negative control (32.00 mg/mL water in acetone) was included to rule out whether the antimicrobial activity was attributed to the solvent. A culture control in TSB was included to ensure the broth supported growth of the reference strains. The samples were then serially diluted down the rows in concentrations of 8.00; 4.00; 2.00; 1.00; 0.50; 0.25; 0.13; and 0.06 mg/mL. After the preparation of an approximate inoculum concentration of 1 × 10 6 colony-forming units (CFU)/mL for each reference strain, 100.00 µL was added to each of the wells. A sterile adhesive sealing film was used to seal the microtiter plate to prevent loss of the samples through evaporation. Incubation of the microtiter plates occurred at 37 • C for 24 h for bacteria and 37 • C for 48 h for the yeast. A volume of 40.00 µL of p-iodonitrotetrazolium violet solution (INT) (Sigma-Aldrich), at a concentration of 0.04 mg/mL, was then added to each well after incubation. The lowest concentration with no colour change was taken as the minimum inhibitory concentration (MIC) for that sample. All samples were tested in triplicate. The average of the samples was calculated and the standard deviation (SD) determined using Microsoft Excel (Microsoft Office Home and Student 2016). Results were considered noteworthy if the MIC value was ≤1.00 mg/mL [5].

Toxicity Studies
The brine shrimp lethality assay [78] was used to determine the toxicity of 21 compounds and six carrier oils alone and in combination. Artificial seawater was prepared by dissolving 16.00 g of Tropic Marine ® sea salt in 500.00 mL of distilled water. This solution was transferred into a bottomless, inverted receptacle. Dried brine shrimp (Artemia franciscana) eggs, from Ocean Nutrition TM , were added to the salt water. Aeration of the water with a rotary pump was included to ensure a high brine shrimp hatch rate. A constant source of light and warmth, from a 220 to 240 V lamp, was used to assist with the hatching process. The eggs were incubated at 25 • C for 24-48 h. For the assay, a 48-well microtiter plate was prepared by adding 400.00 µL of salt water containing 40-60 live brine shrimp to each well. A volume of 400.00 µL of sample was added to each well. For the combinations, a 1:1 ratio of 200 µL each of each sample (carrier oil: compound) was prepared prior to being added to the well containing the shrimp. The assay included a negative, non-toxic control of 32.00 g/L of artificial seawater to ensure the promotion of growth and survival of the brine shrimp. The positive control in the assay consisted of 1.60 mg/mL of potassium dichromate, a highly toxic compound. At 0, 24 and 48 h, the dead brine shrimp were viewed and counted under a light microscope (Olympus) at 40× magnification. A lethal dose of acetic acid (Saarchem; 100% (v/v); 50.00 µL) was added to each well and a final count of dead brine shrimp taken [79]. Then, the percentage mortality was calculated using Equation (1). Biological toxicity was considered for a percentage mortality of 50% or greater [80].
All studies were carried out in triplicate. The average percentage mortality of the brine shrimp was recorded on Microsoft Excel (Microsoft Office Home and Student 2016). % Mortality = Dead shrimp at 24 48 h (before acetic acid) − Dead shrimp (time = 0) Dead shrimp (after acetic acid) × 100 (1)

Interactive Profiles of Combinations
The interactive profiles of the combinations for the antimicrobial and toxicity assays were undertaken, and the fractional inhibitory concentration index (ΣFIC antimicrobial) or the fractional percentage mortality (ΣFPM toxicity) was calculated, respectively, according to Equation (2).
The (a*) represents the essential oil compound in combination and (b*) represents the carrier oil.
The interactive profile was interpreted as follows: an ΣFIC or ΣFPM value of ≤0.5 represented synergy, >0.5-1.0 indicated additive interactions, >1.0-≤4.0 demonstrated indifference, and a value > 4.0 indicated antagonism [81]. For antimicrobial studies, a synergistic combination is regarded as having increased antimicrobial activity and an antagonistic combination is regarded as having decreased antimicrobial activity. Where MIC values of >8.00 mg/mL were determined, they were recorded as 16.00 mg/mL for the purpose of calculating an ΣFIC value. For toxicity studies, synergy is due to a decrease in toxicity of the compounds.

Selectivity Index (SI)
The selectivity index indicates the ratio of toxicity to antimicrobial activity of a sample and was calculated using Equation (3).

Conclusions
This study investigated the antimicrobial and toxicity effects of carrier oils in combination with essential oil compounds. When looking at the antimicrobial activity of 882 combinations, 3% of combinations were synergistic and 4% were antagonistic. The compound thymoquinone and the carrier oil P. armeniaca were present in the majority of the antimicrobial synergistic combinations, and the compound santalol and carrier oil H. perforatum were found in the majority of the antagonistic combinations.
When investigating the toxicity interactions of 105 combinations at 24 h, 10% of the combinations were synergistic, and 77% were antagonistic. When investigating the toxicity of 126 combinations at 48 h, 6% of the combinations were synergistic and 71% were antagonistic. These antagonistic interactions warrant caution when combining equal ratios of compound to carrier oil. The carrier oil C. officinalis was present in the majority of the antagonistic toxicity combinations at 24 h, and the carrier oil P. armeniaca was present in the majority of the synergistic toxicity combinations at 48 h. The selectivity index demonstrated thymoquinone to be the most favorable compound in combination with carrier oils because it was present in the majority of combinations that had an SI value of >4.
Future studies investigating varying ratios may provide a more optimal toxicity profile. It may also be beneficial to investigate the various constituents of the carrier oils themselves, such as the separate free fatty acids and the vitamins, to determine their influence on the essential oil compound toxicity and antimicrobial activity. Nonetheless, this study provides valuable insight into the antimicrobial and toxicity effects of carrier oils when combined with essential oil compounds.