Acute skin irritation , acute and subacute oral toxicity studies of Rosmarinus officinalis essential oils in mice and rabbit

1 College of Medicine, Institute of Medicine and Health Science, Debre-Birhan University, P. O. Box 445, Debre-Birhan, Ethiopia. 2 Department of Plant Biology and Biodiversity Management, College of Natural and Computational Sciences, Addis Ababa University, P. O. Box 34731(Private), Addis Ababa, Ethiopia. 3 Department of Pathology, School of Medicine, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia. 4 Department of Pharmacology, School of Medicine, College of Health Sciences, Addis Ababa University, Addis Ababa, Ethiopia. 5 Department of Chemistry, College of Natural and Computational Sciences, Debre-Birhan University, P. O. Box 445, Debre-Birhan, Ethiopia.


INTRODUCTION
Rosmarinus officinalis (Rosemary) is an evergreen shrub that belongs to the family of Lamiaceae.It is the most common household plants distributed in several parts of the world (Asressu and Tesema, 2014).Rosemary leaves are often employed for various medicinal uses in addition to its role as condiment for flavoring foods.Several ethno-medicinal study reports of Rosemary showed that the plant is widely used for management of headaches, inflammatory diseases, and physical and mental fatigue (Lulekal et al., 2013).Moreover, it has been reported for its use as a stimulant, hepatoprotective and mild analgesic agent (Raškovic et al., 2014).Ethnobotanical investigations on traditional use of R. officinalis in Ankober District, North-Central Ethiopia showed prominent use reports on utilizing leaves of R. officinalis to treat different human and livestock ailments.Moreover, people of the Ankober District frequently use crushed Rosemary leaves in salad, fish and meat as flavor and preventing food poisoning (Lulekal et al., 2013).
Toxicity is the expression of being poisoned that occurs due to the interaction between cells and toxicants (Jothy et al., 2011).For a very long time, several herbal based preparations are supposed to be safe.Yet, many herbal products have been shown to be highly toxic when given either acutely or repeatedly for management of aliments (Prasanth et al., 2015).For instance, ethnobotanical studies in Ethiopia reported traditional herbal preparations side effects such as diarrhea and skin necrosis (Limenih et al., 2015).Hence, it is a timely effort to assess toxicological effects of traditionally used medicinal plant extracts such as that of R. officinalis, preclinically or clinically.This help to identify any potential toxic effect so as to minimize or avoid health-risks.So far, in Ethiopia, studies carried out to evaluate toxicity of essential oils from R. officinalis are lacking.Hence, the present investigation is aimed at investigating acute, subacute and dermatotoxicity effects of R. officinalis leaves essential oil in order to increase the confidence in their safety to humans to treat various ailments.

Collection and identification of plant material
R. officinalis fresh leaves were collected from Ankober Herbal Project Nursery Site which is located in North Shewa Zone, Amhara Regional State, Ethiopia and the identity of the plant specimen was confirmed by a taxonomist at the National Herbarium, College of Natural Science, Addis Ababa University, Ethiopia where a voucher specimen was deposited.

Preparation of essential oil
Thirty grams of shade dried R. officinalis fresh leaves were taken into a 1000-mL round bottomed flask and 300 mL of distilled water was added.Clevenger-type apparatus distilled the mixture for about 3 h to get colorless oil.

Preparation of drug formulation
Ointment (5 and 10% w/w) was formulated for acute dermal toxicity study and skin irritation test, where 5 and 10 g of the extract was incorporated in 100 g (for each) petroleum jelly base.Then physical parameters like physical appearance and homogeneity, viscosity, spreadability and extrudability were evaluated using procedures described elsewhere in Bora et al. (2014) and Nair et al. (2010).

Experimental animals
Female New Zealand rabbits (1.4 to 2.3 kg) and healthy adult Swiss Albino mice of both sexes (10 to 12 weeks of age) were obtained from the Ethiopian Public Health Institute and the School of Pharmacy, AAU animal house, respectively.All the animals were kept at room temperature (25  2°C) in an air conditioned room at 12 h light/dark cycle and acclimatized for 5 days before the study.The animals were provided with water and food pellets ad libtum before and throughout the experimentation period.All the animals were cared for and treated humanely according to the Principles of Laboratory Animal Care (ILAR, 1996).Ethical approval for this study was obtained from Deber Birhan University IRB.

Rabbit skin irritation test
Acute dermal irritation tests were performed using OECD guideline 404 with little modification using two rabbits (OECD, 2002).The fur of the animal was removed by closely trimming the dorsal area of *Corresponding author.E-mail: awolalim@gmail.com.the trunk on different sites 24 h before the test (Figure 1).About 500 mg of 10% of R. officinalis essential oil ointment was applied to two sites and another site was used as a control.Sites were observed critically at 1 h after removal of test substance.The observation was repeated at 24, 48, and 72 h, for days 7 and 15th thereafter.Reactions like erythema and edema were assessed according to the scoring system for skin reactions (Kamkaen et al., 2007;OECD, 2002).

Acute dermal toxicity study
Acute dermal toxicity tests were performed using OECD guideline 434.About 10% of the body surface area fur was shaved 24 h before the study from the dorsal area of the trunk of five female rats showing normal skin texture.A limit test dose of 2000 mg/kg of formulation was applied uniformly over the shaved area for 24 h.Animals were housed individually and general behavioral changes; symptoms of toxicity and mortality were observed after treatment critically for the first 4 h, then over a period of 24 h.At the end of the exposure period, the remaining test substance was removed and the observation was continued daily thereafter for a total of 14 days (OECD, 2004).

Acute oral toxicity study
An acute toxicity study of R. officinalis essential oil was carried out in five female mice using the method of OECD guideline 425 (OECD, 2008).A limit test single dose of 2000 mg/kg of the test sample was given to mice using oral gavage after being deprived of food for 3 h.All the mice were observed for general behavioral changes; symptoms of toxicity and mortality after administration of essential oil for the first 4 h (critically), then over a period of 24 h, and thereafter daily for 14 days.

Sub-acute toxicity study
Sub-acute toxicity study was performed as per the OECD guidelines 407 (OECD, 2008).The treatment (5 male and 5 female) and control (5 male and 5 female) groups orally received R.
officinalis essential oil at the dose of 1000 mg/kg and saline once daily for 28 days, respectively.All mice were observed once in day for any clinical signs of toxicity all the way during the experimental period and the body weight of each mouse was recorded once in a week.At 29th day, mice from both groups were properly anesthetized and blood was collected from a common carotid artery.The serum level of alkaline phosphatase (ALP), serum glutamic-oxaloacetic transaminase (SGOT), and serum glutamicpyruvic transaminase (SGPT) were analyzed.After blood collection, mice from both groups were immediately sacrificed and vital organs of each mouse were examined for the evidence of gross lesions.Liver and kidney were preserved in 10% neutral buffered formalin solution to undertake histopathological examination.Tissues embedded in paraffin wax were sectioned 5 μm thick, stained with haematoxylin and eosin, mounted on glass slides and examined under a standard light microscope (Olaniyan et al., 2016).

Statistical analysis
The data were analyzed by using SPSS version 20 and P < 0.05 was considered as statistically significant.The results were expressed as mean ± standard deviation (SD).

R. officinalis oil ointment formulation
Table 1 shows physical evaluation of R. officinalis essential oil ointment formulation.The ointment formulation was found to be white color, smooth and free from grittiness.The physicochemical properties studied showed satisfactory results for spreadability, extrudability, washability and viscosity.

Rabbit skin irritation test
The effect of the essential oils on dermal irritation is shown in Table 2. From the results of acute dermal irritation study, essential oils appeared to be safe after applying 10% ointment formulation.There was no evidence of any noticeable skin irritation (no erythema and edema) and inflammation in the study period when compared with the control (Figure 1).At 1 h after removal of test substance and thereafter on the skin sites where the test substance was applied, it was found that in all rabbits, erythema and edema score was "0".

Acute dermal toxicity study
No toxic effect was observed on the behavioral response of rats treated with a dose of 2000 mg/kg R. officinalis essential oil.All rats were dosed once and observed for 14 days.Moreover, there were no signs of changes in the behaviour patterns, skin, eyes, salivation, and diarrhea of the rats.Neither mortality nor significant weight loss was also observed.

Acute oral toxicity study
Oral administration of R. officinalis essential oil at the dose of 2000 mg/kg did not show any signs of toxicity and mortality in any of the treated female mice for 14 days during observation period.Hence, the oral lethal dose 50 (LD 50 ) value of R. officinalis essential oil was found to be greater than 2000 mg/kg.

General observation and effects on body weight
Oral administration of R. officinalis essential oil for 28 day caused no noticeable change in the general behavior of the mice and there were no significant changes in body weight or food intake of the mice as compared to the control group.Both the control and treatment groups appeared healthy during the whole study period.There was an increase in body weight of mice in both treatment and control groups.The percentage body weight gain, however, was higher in the control group (17.07%) as compared to treatment groups (7.85%) (Table 3).

The effect of R. officinalis oil on serum biochemical parameters
Sub-acute administration of R. officinalis essential oil effects on biochemical parameters are shown in Table 4. Administration of R. officinalis essential oil for 28 days did not show any significant changes in biochemical parameters such as SGOT, SGPT and ALPL when compared with the control groups.The serum level of SGOT, SGPT and ALP of treatment groups was lower as compared to the control group.Yet, the difference was not statistically significant.

Histopathology
Light microscopic examination of liver and kidney sections of control and treatment groups showed a normal histology (Figures 2 and 3) and absence of any gross pathological lesions after 28 days treatment with 1000 mg/kg dose of R. officinalis essential oil.The result showed normal hepatocellular morphology, normal periportal area with absence of necrosis and inflammation.All kidney sections showed normal glomerular architecture, no vascular necrosis or hyaline changes and unremarkable tubule interstitial paranchyma.
To make sure people using pharmaceutical formulation are safe, assessment of skin irritancy potential to human skin of any chemicals or formulations is compulsory (Kamkaen et al., 2007).The absence of any toxic sign like erythema and edema reactions in present skin irritation test asserts the non-irritant of R. officinalis essential oil ointment.Based on the results, the essential oil primary irritation index was found to be 0 and it was concluded that the essential oil was non-irritating to the skin (PII<0.5).Low camphor content (3.845%) of R. officinalis essential oil might be responsible for absence of skin irritation since camphor is an irritant substance.This finding is in good agreement with that of Hamza et al. (2017), who reported R. officinalis essential oil was non-irritant to the skin though the study was conducted in rat.
Information on target organ toxicity, dosage regimens and observable sign of toxicity that possibly influence life span of experimental animals can be generated by subacute toxicity study (Hilaly et al., 2004).In toxicity study, changes in the body weight serve as a sensitive indicator of general health of experimental animals.There were no significant body weight changes of experimental animals after completing 28 days essential oil administration as compared to the control groups (Table 3).Both treatment and control group animals showed a steady increment in body weight throughout the study period.
Besides body weight, quantification of food and water consumption is necessary during toxicity study of an agent with medicinal value.In this study, R. officinalis essential oil did not affect food and water consumption showing that it did not interfere with the normal digestion and metabolism of animals.For better physiological status of the animal and response to the test substance under investigation, proper intake of supplements is needed (Kumar et al., 2014) Serum biochemicals such as alkaline phosphatase, serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase and creatinine can be used as biomarkers to identify potential toxic effect of drugs and xenobiotics to the vital organ (Brandt et al., 2009;Kumar et al., 2014).Higher blood level of both SGOT and SGPT enzymes are a good indicator of toxicity to the liver parenchymal cells (Kumar et al., 2014).In the present study, there was no statistically meaningful difference in all biochemical parameters measured between the treatment and control groups (Table 4).R. officinalis essential oil did not significantly change the levels of SGOT, SGPT and ALP in experimental animal highlights that it did not affect normal liver function and metabolism of the animals.Histological assessment of the organs (Figures 2 and 3) further confirmed serum biochemical observations.Normal structural features of kidneys histopathological investigation of both treatment and control group propose the normal renal function.Normal architecture of glomeruli and renal tubules portrays the absence of renal toxicity.Liver histopathology of both treatment and control group animals was almost the same with normal morphology of hepatocytes, central vein and portal triads.This finding is in good agreement with that of Nakavuma et al. (2016), who showed lack of gross and microscopic changes in the mice tissues and organs treated with R. officinalis essential oil.

Conclusion
The current finding asserts that R. officinalis essential oil is not toxic and do not produce any adverse effect in the acute and sub-acute oral toxicity investigations.The histology examination revealed no remarkable changes in kidney and liver of the mice in both control and treatment groups.The level of the marker enzymes in the vital organs was also found to be normal.Besides, the skin tolerance test on rabbit showed ointment of R. officinalis essential oil is not irritant and do not show any dermal toxicity.Overall, it can be concluded that the R. officinalis essential oil is well tolerated in daily dose at 1000 mg/kg for a period of 28 days and safe for traditional use and as food additive.

Figure 1 .
Figure 1.Photograph of skin of rabbit before application test ointment (A) and 1hr after removal of R. officinalis essential oils ointment formulation (B).

Figure 2 .Figure 3 .
Figure 2. Liver histology of control and R. officinalis essential oil treated animals.(A) Liver section of control animals revealed normal architecture and hepatic cells; (B) Liver section of R. officinalis essential oil (1000 mg/kg) treated animals exhibited normal architecture of hepatocytes and hepatic cells after 28 days of treatment.

Table 1 .
Physical evaluation of 10% R. officinalis essential oil ointment formulation.

Table 2 .
Score of erythema and edema after application of test materials on rabbit skin.
Score for skin reaction of 10% R. officinalis essential oil ointment (three treatment site) at different time interval after removing test ointment.Primary Irritation Index (PII) = 0/3, PII=0; Category of irritation based on PII is Negligible.Con: Control; Treat: treatment.

Table 3 .
Effects of R. officinalis essential oil on body weight of mice treated orally for 28 days.

Table 4 .
Biochemical parameters of mice treated orally with R. officinalis oil for 21 days.