ABSTRACTrnBackground:In Ethiopian traditional medicine, the aerial part of Thymus schimperi is widelyrnused to treat diseases such as gonorrhea, cough, liver disease, kidney disease, hypertension,rnstomach pain, and fungal skin infections. In addition, theyhave been used as vegetables to flavorrna broad variety of food products. However, there is an insufficient investigation of the toxicrneffect of T. schimperi. The aim of this study was, therefore, to evaluate the acute, sub-acute,rndevelopmental, and in-silico toxicity of the essential oil,as well as the developmental toxicity ofrnthe aqueous crude extract of T. schimperi on the Wistar albino rats.rnMethod: The aqueous extracts of T. schimperi leaves were prepared.Essential oil of the aerialrnpart of T. schimperiwas extracted by hydrodistillation and was analyzed by GC-MS. The oil wasrnsubjected to toxicity studies. In the acute toxicity study, rats were randomly divided into sevenrngroups (n=5). The control group received the vehicle (distilled water and 2% tween 80)whereasrnthe experimental groups received single doses of 300, 600, 900, 1200, 1500, and 2 000 mg/kg ofrnvehicle dissolved essential oil. In the sub-acute toxicity study, rats were randomly divided intornfour groups (n=10). The control group received the vehicle whereas the experimental groupsrnreceived vehicle-dissolved doses of 65 mg/kg, 130 mg/kg, and 260 mg/kg of oil orally for 28rndays. At the end of the experiment, blood samples were collected for hematology and clinicalrnchemistry evaluation. Gross pathology and histopathology of the liver and the kidneys were alsornevaluated. For the in-silico toxicity study, PubChem CID numbers of GC-MS identifiedrnbioactive compounds in essential oils of T. schimperi have been obtained from PubChem.rnChemdraw (8.0) was used to construct the two-dimensional structure of the compounds. ThernSwiss ADMET web tool was used to convert the two-dimensional structures into a simplifiedrnmolecular-input line input system (SMILES). Furthermore, the toxicity parameters werernpredicted via Protox II, vNN, and ADMET servers. For aqueous and essential oil extractsrndevelopmental toxicity experiments, five groups of Wistar albino rats, each consisting of tenrnpregnant rats, were used as experimental animals. For the aqueous crude extract developmentalrntoxicity study, the rats in groups III-V were given 500 mg/kg, 1000 mg/kg, and 2000 mg/kgrnextract of T.schimper, respectively. On the other hand, in the essential oil developmental toxicityrnstudy, the doses 65 mg/kg, 130mg/kg, and 260 mg/kg of the essential extract of T. schimperirnwere administered forIII-V groups, respectively. Group I and II were negative and ad libitumrncontrols for both experiments. Similarly, Embryos and fetuses were revealed on days 12 and 20rnof gestation, respectively. The embryos were examined for developmental delays or growthrnretardation. Gross external, skeletal, and visceral anomalies in the fetuses were examined.rnHistopathological examination was carried out on the placenta from both the treatment andrncontrol groups.rnResults: In this study, the LDrnof the essential oil of T. schimperi was found to be 1284.2rnmg/kg. According to the World Health Organization, the oil is classified as moderatelyrnhazardous in its oral administration. In the subacute toxicity study, rats showed no significantrnchanges in behavioral indices, gross pathology, body weight, biochemical, and in mostrnhematological parameters. However, hematological profiles showed a significant decrement inrn50WBC counts and a significant increment of MCV in high dose (260 mg/kg) groups as comparedrnto the control group. Furthermore, no significant differences were observed between the controlrnand essential oil-treated groups, observed in the gross and histopathology of the liver and thernkidneys.rnIn the in-silico toxicity study, all compounds derived from essential oil showed no cardiacrntoxicity (h-ERG Blocker), AMES (Ames Mutagenicity), and cytotoxicity via Pro Tox II,rnADMET, and vNN-ADMET toxicity predictors. However, by using these servers, from the totalrn57 compounds, around 21% showed carcinogenicity, 8.8% showed hepatotoxicity, 3.5% causedrndrug-induced liver injury, 3.5% showed immunotoxicity, and only 1.75 % were potentially toxicrnto the mitochondrial membrane.rnIn the aqueous crude extract developmental toxicity study, on embryo day 12, the number ofrnsomites and the morphological scores in the high-dose treatment group were significantly lowerrnthan the control groups. Similarly, the number of implantation sites, fetal weight, fetal resorption,rnCRL, and placental weightwere also significantly lower in the high dose (2000 mg/kg)treatmentrngroup. The mean numbers of implantation sites in the pair-fed control group and the highrndose(2000 mg/kg)group were 11.1 Â± 0.76 and 8.01 Â± 0.45, respectively. Similarly, in the middlerndose (1000 mg/kg)and high dose (2000 mg/kg)groups, the developments of the otic andrnolfactory systems were significantly delayed. Furthermore, in the high dose group (2000 mg/kg),rnthe developmental score of optic system, the number of branchial bars, and the maxillary andrnmandibular processes were significantly lower than the control groups. Treatment with thernaqueous extract of the T.schimpericaused no skeletal or soft tissue malformations. In an essentialrnoil developmental toxicity study, the developmental socres of fetal resorptions, crown-rumprnlength, the number of somites, and morphological scores were significantly lower in 12-day-oldrnrat embryos treated with 260 mg/kg of the extract. There was also a significant delay in therndevelopments of the otic system, olfactory system, and a reduction in the number of branchialrnbars in day-12 embryos treated with 260 mg/kg of the oil.However, external morphologicalrnexaminations of rat fetuses revealed no detectable structural abnormalities. The fetal skull,rnvertebrae, hyoid, forelimb, and hindlimb ossification centers did not differ significantly across allrngroups. Moreover, treatment with the essential oil caused no skeletal or soft tissuernmalformations. Although the difference was not statistically significant, fetuses of high-doserntreated rats had a reduced number of ossification centers in the caudal vertebrae and hind limprnphalanges.There were no significant histopathological changes in placentas in either the crudernaqueous extract or the essential oil experiments. Although the difference was not statisticallyrnsignificant, placentas from high-dose essential oil treatment rats had increased decidual cysticrndegeneration, thrombosis in the intervillous spaces, and decidual cellular apoptosis. Similarly, inrnthe essential oil experiment, capillary dilation and terminal villi proliferation increased dosedependently.rnConclusion:rnFrom this study, oral administration of the essential oil T. shimperi up to a dose ofrn130 mg/kg is not harmful. However, in the high-dose (260 mg/kg) group, the WBC count wasrnsignificantly decreased and the MCV was significantly increased. In the in-silico toxicity study,most of the components of the oil were found to be nontoxic although a few of the compoundsrnshowed carcinogenicity, hepatotoxicity,immunotoxicity and mitochondrial membrane potentialrntoxicity.The crude aqueous and essential oil extracts of T. shimperi at high doses have arndetrimental effect on the development of rat embryos and fetuses. Its developmental toxicity isrnevidenced by significant delays in fetal and embryonic development, a decrease in the number ofrnimplantation sites, and an increase in fetal resorption. Furthermore, administration of the aqueousrncrude and essential oil extracts in higher doses resulted in a significant decrease in placentarnweight, and litter weight. It is, therefore, essential to conducting chronic toxicity of the essentialrnoil as well as its components which showed toxicity in the in- silico study before usingrnpreparations containing T. schimperi essential oil as drugs. In addition, the present studyrnprovided evidence that using the T.schimperi extracts in a high dose could affect the developingrnembryo and fetus. Thus, it is recommended to discourage the use of crude and essential oilrnextracts in high doses.