Melaleuca alternifolia, ext.

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050, "Repeated Dose 28-Day Oral Toxicity Study in Rodents".

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Lot/batch No.of test material: H302
- Expiration date of the lot/batch: June 15, 2018
- Purity: 100%
- Storage condition of test material: Ambient

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CRL Sprague-Dawley CD IGS rats

Details on species / strain selection:

The Sprague-Dawley rat is the system of choice because, historically, it has been a preferred and commonly used species for oral toxicity tests. The current state of scientific knowledge does not provide acceptable alternatives to the use of live animals to accomplish the objective of this study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Inc.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Eight weeks
- Weight at study initiation: 226-260 g (males) and 167-196 g (females). The weight variation did not exceed ± 20% of the mean weight for each sex.
- Housing: Animals were individually housed in suspended stainless steel cages which conform to the size recommendations. Litter paper placed beneath the cage was changed at least three times per week.
- Diet: 2016 Certified Envigo Teklad Global Rodent Diet (Envigo Teklad, Inc.) was stored in a dedicated temperature and humidity monitored feed storage site and was available ad libitum during acclimation and throughout the study.
- Water: Filtered tap water was available ad libitum from an automatic watering access system.
- Acclimation period: Eight days prior to testing.
- Temperature (°C): 19-23
- Humidity (%): 41-60
- Photoperiod: 12 hour light/dark cycle

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected by the Sponsor. This route of administration is recommended in the guidelines, and is a potential route of human exposure.

Vehicle:

corn oil

Details on oral exposure:

- Test substance preparation: The test substance was mixed weight to volume (w/v) in corn oil. Fresh formulations were prepared daily. The formulations were stirred at ambient temperature until a visually homogeneous solution was achieved.
- Dose calculations: Individual doses were calculated based on the most recent weekly body weights and were adjusted each week to maintain the targeted dose level for all rats (i.e. mg/kg/day). All doses were administered volumetrically at 5 mL/kg. The control group received vehicle (corn oil) only, at the same dose volume as the test animals.
- Dosing: Each animal was dosed by oral intubation using a stainless steel ball-tipped gavage needle attached to an appropriate syringe. Dose administration was daily (7 days/week) for a period of at least 28 days. The dose solution was maintained on a magnetic stir plate during administration. The first day of dose administration was considered Day 1 of the study. Dosing was at approximately the same time each day (±2 hours) with an exception on the day(s) hematology and/or clinical chemistry samples were collected.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Sampling: The neat test substance and dose preparations were sampled in duplicate.
- Test substance stability: The test substance was expected to be stable over the course of the study under the conditions of storage. A sample of the neat test substance was collected at the beginning and end of the in-life phase. Initial and final results were compared to demonstrate test substance stability.
- Dose preparation homogeneity: On the first day of dosing, formulations of each concentration were prepared according to the procedures as was used on test. Samples from these preparations were collected from the top, middle, and bottom of each concentration of test substance that was prepared in the control vehicle. Samples of the vehicle control were sampled from the middle of the container only. Samples were analyzed to verify the preparations as homogeneous and of accurate concentration throughout the study.
- Concentration verification: The dose preparations were sampled at the beginning (as part of the homogeneity assessment), near the middle, and again at the end of the study for verification of dose concentration. Samples were collected from each concentration of test substance that was prepared in the vehicle, and one sample from the vehicle control was collected.
- Sample preservation: Upon sampling, dose preparations and neat test substance were stored frozen. Samples were considered stable from the point at which they were frozen. Samples were then sent for analysis.
- Method validation: Prior to sample analysis, the suitability of the method was demonstrated. Method validation included, but was not limited to determination of linearity, precision and accuracy.
- Reference substance: Neat test substance sample served as the reference standard.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Daily (7 days/week).

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control group (Group 1). Dose volume 5 mL/kg/day. Test substance concentration: 0 mg/lL.

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Remarks:

Low dose group (Group 2). Dose volume: 5 mL/kg/day. Test substance concentration: 1 mg/mL.

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Remarks:

Intermediate dose group (Group 3). Dose volume: 5 mL/kg/day. Test substance concentration: 3 mg/mL.

Dose / conc.:

45 mg/kg bw/day (actual dose received)

Remarks:

High dose group (Group 4). Dose volume: 5 mL/kg/day. Test substance concentration: 9 mg/mL.

No. of animals per sex per dose:

5 male and 5 female animals per test group (40 in total).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected by the Sponsor in consultation with the Study Director, based on the results of a previous range-finding/toxicity study.
- Rationale for animal assignment: Animals were randomly assigned, stratified by body weight, to test groups.

Observations and examinations performed and frequency:

- Cage side observations: Cage side observations of all animals were performed daily during the study. All findings were recorded.

- Detailed clinical observations: Prior to the first treatment with the test substance, and weekly thereafter, a detailed clinical observation was conducted while handling the animal, generally occurring on days that the animals were weighed and food consumption measurements taken. Potential signs noted included, but were not limited to: changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Likewise, changes in gait, posture, and response to handling, as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded. The date and clock time of all observations and/or mortality checks were recorded.

- Body weight: Individual body weights were recorded at least two times during acclimation. Test animals were weighed on Day 1 (prior to study start) and approximately weekly thereafter (intervals of 7 days ± 1). The animals were also weighed immediately prior to sacrifice in order to calculate organ-to-body weight ratios. Body weight gain was calculated for selected intervals and for the study overall.

- Food consumption: Individual food consumption was measured and recorded to coincide with body weight measurements.

- Food efficiency: Food efficiency was calculated and reported.

- Clinical pathology: Clinical pathology was performed on all main test animals for blood chemistry and hematology of the terminal sacrifice animals once toward the end of the dosing phase of the study. The animals were fasted overnight prior to blood collection. Blood samples for hematology (except coagulation samples) and clinical chemistry were collected during approximately Week 4 of the test period. Blood samples used to determine the prothrombin time and activated partial thromboplastin time (coagulation) were collected at terminal sacrifice. A second blood sample was retained during the exsanguination procedure for future possible evaluation, if treatment-related effects were identified.

- Haematology: The examined parameters included erythrocyte count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width (RDW), absolute reticulocyte count (ARET), platelet count (PLT), total white blood cell (WBC) and differential leukocyte count. Mean corpuscular hemoglobin concentration (MCHC) was calculated. In addition, separate, blood smears, stained with New Methylene Blue or Wright-Giemsa stain, were prepared from each animal undergoing hematological evaluation.

- Coagulation: Coagulation included prothrombin time (PT) and activated partial thromboplastin time (APTT).

- Clinical chemistry: The examined parameters included serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALKP), total bilirubin (BILI), urea nitrogen (BUN), blood creatinine (CREA), total cholesterol (CHOL), triglycerides (TRIG), fasting glucose (GLUC), total serum protein (TP), albumin (ALB), globulin (GLOB), calcium (CALC), inorganic phosphorus (IPHS), sodium (NA), potassium (K) and chloride (CL). Any remaining serum samples were maintained frozen at approximately -80°C and discarded upon approval of the Sponsor at finalization.

- Other: All animals were observed at least twice daily for viability.

Sacrifice and pathology:

- Scheduled sacrifice: At terminal sacrifice, all animals in the study were subjected to a full necropsy, which included examination of the external surface of the body, all orifices, and the thoracic, abdominal and cranial cavities and their contents. All gross lesions were recorded. The following tissues (of all animals) were weighed wet: adrenals (combined), brain, epididymides (combined), heart, kidneys (combined), liver, testes (combined), ovaries with oviducts (combined), spleen, thymus and uterus.

The following organs and tissues from all animals were preserved for possible future histopathological examination: accessory genital organs (prostate and seminal vesicles), adrenals, all gross lesions, aorta, bone (femur), bone marrow (from femur and sternum), brain (3 sections including medulla/pons, cerebellar, and cerebral cortex), cecum, cervix, colon, duodenum, esophagus, Harderian gland, heart, ileum with Peyer’s patches, jejunum, kidneys, larynx, liver, lungs, lymph node mandibular, lymph node mesenteric, mammary gland, nasal turbinates, nose, ovaries, oviducts, pancreas, parathyroid, peripheral nerve (sciatic), pharynx, pituitary gland, rectum, salivary glands (sublingual, submandibular, and parotid), skeletal muscle, skin, spinal cord (3 levels: cervical, mid-thoracic, and lumbar), spleen, sternum, stomach, thymus, thyroid, trachea, urinary bladder, uterus, vagina, eyes, epididymides, optic nerve and testes.

- Histopathology: Histological examination was performed on the preserved organs and tissues of the animals from both the control and high dose groups (Groups 1 and 4, respectively). In addition, gross lesions of potential toxicological significance noted in any test groups at the time of terminal sacrifice were also examined.

Other examinations:

- Functional observation battery: Once towards the end of the exposure period (4th week), a functional observation battery (FOB) was performed on all surviving animals. Each rat was evaluated during handling and while in an open field for excitability, autonomic function, gait, and sensorimotor coordination (open field and manipulative evaluations), reactivity and sensitivity (elicited behavior) and other abnormal clinical signs, including but not limited to convulsions, tremors, unusual or bizarre behavior, emaciation, dehydration, and general appearance. The rats were observed in random order and without the observer having knowledge of the treatment group.
- Motor activity: Motor activity (MA) was also evaluated on all surviving animals. This assessment coincided with FOB assessments. Activity was monitored using an automated Photobeam Activity System® (San Diego Instruments, Inc.). The system can monitor up to twenty-one animals during one session. An equal number of animals assigned to motor activity assessment from each dose group was evaluated in each session, when applicable. Each animal was placed into a polycarbonate solid bottom cage and the evaluation phase began immediately for that animal. Each animal was evaluated for a single one-hour phase, with photobeam counts accumulated over six 10-minute intervals.

Statistics:

Statistical analysis was performed on all data collected during the in-life phase of the study as well as organ weight data. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p<0.05. Male and female rats were evaluated separately.

- In-life and organ weight data: Mean and standard deviations were calculated for all quantitative data. If warranted by sufficient group sizes, data within groups were evaluated for homogeneity of variances and normality by Bartlett’s test (Bartlett, 1937). Where Bartlett’s test indicated homogeneous variances, treated and control groups were compared using a one-way analysis of variance (ANOVA). When one-way analysis of variance was significant, a comparison of the treated groups to control by Dunnett’s test (Dunnett, 1964, 1980) for multiple comparisons was performed. Where variances were considered significantly different by Bartlett’s test, groups were compared using a non-parametric method (Kruskal-Wallis non-parametric analysis of variance; Kruskal and Wallis, 1952). When non-parametric analysis of variance was significant, comparison of treated groups to control was performed using Dunn’s test (Dunn, 1964). Statistical analysis was performed on all quantitative data for in-life and organ weight parameters using Provantis version 9, Tables and Statistics, Instem LSS, Staffordshire UK.

- Clinical pathology: See 'Any other information on materials and methods' for details on the statistical methods used for clinical pathology.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical observations attributed to administration of the test substance (see attached Table 2 and Table 3).
- Males: Incidental, in-life clinical observations included slight to moderate alopecia on the left/right forelimb/forepaw of 1/5 Group 2, 1/5 Group 3, and 1/5 Group 4 animals. Corresponding detailed clinical observations included hair loss in 1/5 Group 2, 1/5 Group 3, and 1/5 Group 4 animals. Additional detailed clinical observations included a broken toenail on the left hindpaw of 1/5 Group 1 animals.
- Females: Incidental in-life clinical observations included slight alopecia on the left/right forepaw/forelimb of 1/5 Group 1 and 1/5 Group 4 animals. Corresponding detailed clinical observations included hair loss in 1/5 Group 1 and 1/5 Group 4 animals.

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related mortalities during the course of the 28-day study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes in body weight or body weight gain attributable to administration of the test substance (see attached Table 6 and Table 7). Mean weekly body weights and mean daily body weight gain for male and female rats in Groups 2-4 were comparable to control Group 1 values throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes in food consumption attributable to administration of the test substance (see attached Table 8). Mean daily food consumption for male and female rats in Groups 2-4 were comparable to control Group 1 values throughout the study.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no changes in food efficiency attributable to administration of the test substance (see attached Table 9). Mean food efficiency for male and female rats in Groups 2-4 were comparable to control Group 1 values throughout the study.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in hematology parameters for male or female rats on Day 27 (see attached Table 10). There were no test substance-related changes in coagulation parameters for male or female rats on Day 29 (see attached Table 11).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance related changes in serum chemistry parameters for male or female rats on Day 27 (see attached Table 12). Other differences in serum chemistry parameters that were statistically significant are listed below. These were observed in a non-dose dependent manner, are within the historical data base range, are within expected biological variation and are not toxicologically relevant:
- Increased glucose levels for Group 2 males; Increased bilirubin levels for Group 3 females; Increased inorganic phosphorus for Group 2 females.

Urinalysis findings:

not examined

Description (incidence and severity):

.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional behavioral tests performed near termination (Day 26) of the study were comparable between the test and control groups of male and female rats. There were no observations attributed to administration of the test substance (see attached Table 4). Incidental findings in males included hair loss in 1/5 Group 2, 1/5 Group 3 and 1/5 Group 4 animals. In females, incidental finding s included hair loss in 1/5 Group 1 and 1/5 Group 4 animals.
There were no changes in motor activity attributable to administration of the test substance (see attached Table 5).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no changes in absolute organ weights or organ weights relative-to-body weights attributable to administration of the test substance (see attached Tables 14, 15 and 16).
- Male: Mean absolute and relative-to-body organ weights for rats in Groups 2-4 were comparable to control Group 1 values. Mean relative-to-brain organ weights for Groups 2-4 were generally comparable to control Group 1 values with the exception of a significant increase (p<0.05) in mean liver-to-brain weights for Group 3 animals that is considered not to be toxicologically relevant
- Female: Mean absolute and relative organ weights for rats in Groups 2-4 were comparable to control Group 1 values.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The uterus was noted as “fluid filled” at necropsy in 1 out of 5 females at 0 mg/kg/day Tea Tree Oil, in 0 out of 5 females at 5 mg/kg/day Tea Tree Oil, in 1 out of 5 females at 15 mg/kg/day Tea Tree Oil, and in 1 out of 5 females at 45 mg/kg/day Tea Tree Oil. Microscopically, fluid filled uteri corresponded to lumenal dilation of the uterus that was attributable to variation in the estrous cycle in individual animals and was not associated with the test substance (see attached Table 13).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic observations: At the Day 29 time point, there were no microscopic findings related to the administration of the test substance. The microscopic findings observed were sporadic and were incidental as can be observed in the age and strain of rats used in this study.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

45 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Critical effects observed:

no

Test Substance and Dose Preparation Analysis

The neat test substance was stable under the conditions of storage over the course of this study. Based on the overall neat test substance stability, homogeneity, and concentration verification analysis, animals were generally considered to have received target concentrations of Tea Tree Oil or higher. An exception occurred on Day 14, when the Group 2 dosing solution assayed at 0% of target; as this group does not set the NOAEL, this exception is interpreted to have no impact.

Analysis of Tea Tree Oil - Neat Test Substance

The test substance was found to be stable under the conditions of storage over the course of this study. Results of the stability analysis of the neat Tea Tree Oil were 100.7% of the target concentration on Day 1 (initial) and 99.7% on Day 28 (final). The difference in the neat test substance concentration over the course of the study was -0.95% and the overall test substance stability was determined to be 99.1% (see attached Table 1A).

Homogeneity

Homogeneity analysis of the dose preparations resulted in a relative standard deviation (RSD) of 21.5, 1.9, and 0.7% for target concentrations of 1, 3, and 9 mg/mL, respectively. Average percent of target concentrations in the top, middle, and bottom samples were 167.4, 102.6, and 98.8%, respectively. Results observed on Group 2 samples have no toxicological impact because animals received at least the targeted dose levels. An error of sampling of the control dosing preparation resulted in recovery of test substance from the control sample from Study Day 1; since no effects were observed for this group throughout the study, there is no impact on the overall study (see attached Table 1B).

Concentration Verification

Concentration verification analysis samples were collected on the day of preparation for initial (Day 1, as part of the homogeneity assessment), mid (Day 14) and final preparations (Day 28). The Day 1 samples averaged 167.4, 102.6, and 98.8%, the Day 14 samples averaged 0, 78.2, and 109.1% , and the Day 28 samples averaged 94.2, 95.3, and 83.2%, the target concentrations of 1, 3, and 9 mg/mL, respectively. On Day 14, Group 2 dosing solution assayed at 0% of target; as this group does not set the NOAEL, this exception is interpreted to have no impact (see attached Table 1C).

Conclusions:

Under the conditions of the study and based on the toxicological endpoints evaluated, the no adverse effect level (NOAEL) for administration of Tea tree oil was determined to be 45 mg/kg/day, for both male and female Sprague Dawley rats.

Executive summary:

A GLP compliant, 28 -day oral toxicity study was conducted in CRL: Sprague Dawley CD IGS rats to determine the tolerability and general toxicity of tea tree oil in accordance with OECD guideline 407. Forty healthy rats (20 males and 20 females) were selected for the test and distributed into four groups (5/sex/group, 1 vehicle control and 3 test substance groups). Dose levels of 0 mg/kg/day for vehicle control (corn oil), 5, 15, and 45 mg/kg/day were selected for test Groups 1 -4, respectively.

An appropriate amount of the vehicle control or diluted test substance was administered daily via oral gavage to each rat for 28 consecutive days. The test substance was administered at concentrations of 1 mg/mL (low dose), 3 mg/mL (intermediate dose), and 9 mg/mL (high dose) w/v solutions in corn oil. The vehicle (corn oil) and test substance preparations were administered at 5 mL/kg/day. Each dose was prepared daily.

The neat test substance was determined to be stable under the conditions of storage over the course of this study. Based on the overall neat test substance stability, homogeneity, and concentration verification analysis, animals received target concentrations of Tea Tree Oil or higher.

The animals were observed for viability, signs of gross toxicity, and behavioral changes at least once daily during the study and weekly for a battery of detailed clinical observations. Body weight and food consumption measurements were collected throughout the study. Mean body weight gain and mean food efficiency were calculated. Gross necropsies and histological evaluation of selected organs and tissues were performed on selected study animals.

There were no test substance-related mortalities or changes in clinical or detailed observations, functional observation battery, motor activity body weight, body weight gain, food consumption, or food efficiency during the study. There were no macroscopic, microscopic or organ weight findings attributed to test substance administration. The NOAEL was determined to be 45 mg/kg/day for both male and female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

45 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

This study has been assigned a reliability of 1 and meets the information requirements of REACH.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

A 28 day oral toxicity study was conducted by gavage in Sprague-Dawley rats (Koetzner, 2017).  No adverse effects attributable to the test substance were observed in animals dosed at up to 45 mg/kg bw/day.  

As no significant adverse effects were observed at the CLP guidance values applicable to 28-day studies for classification as STOT RE, it is concluded that tea tree oil does not meet the criteria for classification on the basis of repeat dose toxicity.