Eucalyptus maculata citriodora, ext.

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15-12-2016 to 16-02-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(R)-3,7-dimethyloct-6-enal

EC Number:

219-194-5

EC Name:

(R)-3,7-dimethyloct-6-enal

Cas Number:

2385-77-5

Molecular formula:

C10H18O

IUPAC Name:

3,7-dimethyloct-6-enal

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Obtained from sponsor
- Expiration date of the lot/batch: 31-12-2017
- Purity test date: 9-8-2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was formulated daily, using powdered diet, by initial preparation of a premix followed by dilution with further quantities of diet and mixing.

FORM AS APPLIED IN THE TEST
Mixed into powdered rodent diet

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat is the species and strain of choice because it is accepted by regulatory authorities, recognized as appropriate for general and reproduction toxicity studies and there is ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 203-228 g for males and 177-206 g for females
- Housing:

From arrival to pairing: animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating: animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
After mating: the males were re-caged as they were before mating.The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm). Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week.

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks for main groups and 3 weeks for recovery groups

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From experimental start date: 15 December 2016 To: 16 February 2017

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as it is a possible route of exposure of the test item in man.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The test item was formulated daily, using powdered diet, by initial preparation of a premix followed by dilution with further quantities of diet and mixing, at fixed concentrations of 1000, 3000 and 10000 ppm. The formulations were prepared separately for each group. Concentrations were calculated and expressed in terms of test item as supplied.

- DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with (Type of food): Powdered rodent diet [4RF21Mucedola s.r.l, SettimoMilanese (MI), Italy].

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The proposed formulation procedure for the test item mixed with the diet was checked in the range of 1000 to 10000 ppm by chemical analysis (concentration and homogeneity) during the pre-treatment period, to confirm that the method was suitable.
Stability after 28 hours at room temperature (range of 1000 and 10000 ppm) was also verified. Samples of the diet mix prepared during the study were also analysed to check the concentration and homogeneity (two occasions during the study, first and last week of treatment). Results of all analyses were within the acceptability limits (80-120% for concentration and CV<10% for homogeneity). Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. A1357).

Duration of treatment / exposure:

Males: 2 consecutive weeks before pairing, during pairing and followed by 2 consequtive weekes up to the day of necropsy.
Females: 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods up until Day 4 post partum (the day of sacrifice)
Recovery groups: Animals had access to their appropriate diets (treated or control diet) depending on treatment group for 4 consecutive weeks. Untreated diet was given during the recovery period.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Included in both main and recovery group

Doses / concentrations 2

Dose / conc.:

1 000 ppm

Remarks:

Intended dose level: 100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

3 000 ppm

Remarks:

Intended dose level: 300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

10 000 ppm

Remarks:

Intended dose level: 1000 mg/kg bw/day / Included in both main and recovery group

No. of animals per sex per dose:

10 in the main groups
5 in the recovery groups

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Nominal dose levels were selected in agreement with the sponsor, based on a previous preliminary, non GLP compliant study (RTC Study No.: E0094).
- Rationale for animal assignment (if not random): The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
- Post-exposure recovery period: 2 weeks

Positive control:

Not included

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily during the study, each animal was observed and any clinical signs recorded. Furthermore, animals were checked for mortality early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Weighed weekly from allocation to termination
Females: Were weighed weekly from allocation to positive identification of mating and on days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.
Recovery: Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: as a part of the sacrificial procedure, and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters examined:
Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count - Neutrophils - Lymphocytes - Eosinophils - Basophils - Monocytes - Large unstained cells, Platelets, Prothrombin time, Coagulation, Activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as a part of the sacrificial procedure, and at the end of the recovery period.
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters examined:
Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Inorganic phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride.

URINALYSIS: Yes (in 5 males randomly selected)
- Time schedule for collection of urine: as a part of the sacrificial procedure, and at the end of the recovery period.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined.
Appearance, Volume, Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood, Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before commencement of treatment and at least once a week thereafter.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity, grip strength andmotor activity

OTHER: Vaginal smears and oestrus cycle - Parturition and gestation length

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All females were examined for the following:
1. external and internal abnormalities;
2. number of visible implantation sites (pregnant animals);
3. number of corpora lutea (pregnant animals).

HISTOPATHOLOGY: Yes

ORGANS/TISSUES (incl. histologic examinations):
Adrenal glands
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Clitorids
Colon
Duodenum
Epididymides
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Mammary area
Nasal cavity*
Oesophagus*
Ovaries with Oviducts
Parathyroid glands
Pituitary gland
Penis
Prostate gland
Rectum
Sciatic nerve
Seminal vesicles with coagulation glands
Spinal column*
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach
Testes
Thymus (where present)
Thyroid
Trachea
Urinary bladder
Uterus – cervix
Vagina
*Not examined histologically since no signs of toxicity or target organ involvement were noted

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant clinical signs were observed throughout the study in treated animals of both sexes. During mating phase, in one male of the low dose group and in one male of the high dose group hairloss and/or scabs were noted.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight of treated males did not show differences of toxicological significance throughout the study, when compared to the control group. Body weight gain was decreased compared to controls in all males (statistically significant only for the high dose group), before pairing. At the end of the mating phase, adose-related decrease was also noted in all treated groups (not significant). Body weight of females, before pairing, was statistically significant decreased in high dose group. During the gestation and post partum periods, body weight of high dose females was significantly lower than control group. Body weight gain in all treated females, before pairing, was decreased compared to controls, with dose-relation (not significant). Decrease in body weight gain was still observed in the high dose females during the gestation period (significant in the high dose group at day 14). Means of body weight were comparable between control and treated groups both in males and females throughout the treatment phase.
Body weight gain in treated males and females decreased during treatment period (not significant). During the recovery period, an increment in bodyweight gain was noted in males and females when compared with control group (significant). Slight reduction noted in terminal body weight of treated male and female animals (ranging from -3% to -10%) was not considered toxicologically relevant due to the very slight severity even if statistically significant for females of the high dose group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Main groups
The food consumption was moderately reduced, especially in the first day of administration, in both sexes of the high dose group. In the low and mid-dose groups, the food consumption of both sexes was slightly reduced when compared to control group. Generally, the food consumption in the high dose group remained reduced when compared to control for the entire period of treatment. In addition, in females of the high dose group during the post coitum period, the food consumption was statistically significant reduced when compared to the control group.

Recovery groups
During treatment, the food consumption was generally slightly reduced in both sexes especially in the first days of administration. During recovery periods, the food intake become comparable with the control group or, for males, the food intake was higher than controls. No toxicological relevance was attributed to the slight reduction noted in food consumption.

Compound intake
Using fixed dietary inclusion levels of the test item, the mean achieved dosages for the dosing period were found to be comparable with the expected ones (approximately 1000, 3000 and 10000 ppm) for main group males, while slightly lower for main group females and recovery animals. This discrepancy is due to a lower than expected food consumption. The achieved dosages in mg/kg/day, for each group, are presented in the tables.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Main groups
One male dosed at 10000 ppm showed severe leucocytosis, mainly due to augmented neutrophils (10.2 fold above mean control data). Slight neutrophilia was recorded in other two males from the same treated group. Statistically significant decrease of prothrombin time and activated thromboplastin time was recorded in males receiving 10000 ppm (8% for both parameters). In addition, statistically significant increases of erythrocytes, haemoglobin and mean corpuscular volume were observed in females receiving 10000 ppm. Due to the slight severity (4% to 11%) and the direction, these findings were considered of no toxicological relevance. Activated thromboplastin time was decreased in females receiving 1000 mg/kg bw/day (17%). Due to the slight severity and the direction, changes were considered not adverse, even though they could be treatment-related.

Recovery groups
Changes recorded during the dosing phase were no longer observed. The slight increases of lymphocytes and large unstained cells recorded in treated females were not observed during the dosing phase, therefore they were considered unrelated to treatment. No differences in coagulation were recorded between control and treated animals, confirming reversibility.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups
Statistically significant fluctuations of some biochemical parameters were recorded, such as: increase of calcium in males dosed at 10000 ppm (4%), decrease of sodium in those receiving 3000 ppm (1%), decrease of alkaline phosphatase in females treated at 3000 ppm (38%), decrease of creatinine and phosphorus in females receiving 3000 and 10000 ppm (approximately 19% for creatinine and 29% for phosphorus). Due to the minimal severity, the direction of changes and/or the absence of dose-relation, the above findings were considered of no toxicological relevance.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Main groups
The major changes observed in treated males were an increase in absolute and relative kidneys weights in mid- and high dose groups (absolute: +7%, +18%; relative: +13%, +25%). These changes in kidneys weights could be related to the hyaline droplet accumulation observed at histopathological examination performed in high dose male animals. In treated females a statistically significant decrease was seen in absolute ovaries weight of the high dose group (-16% ) which could be related to the reduction in number of corpora lutea noted in these animals.

Some other statistically significant differences were noted in the absolute and/or relative organ weights, such as:
– Increase in relative epididymides (+9%) and testes (+17%) weight in high dose males.
– Decrease in absolute heart (-10%), spleen (-13%), thymus (-24%) and uterus (-17%) weight in high dose females.
These alterations were not accompanied by histopathological findings and were therefore considered unrelated to treatment.

Recovery groups
After 2 weeks of recovery period, terminal body weight of treated animals was comparable to the control group. No relevant differences were observed in the organs of treated animals when compared to controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross lesions such as reduced size of thymus in two high dose females, swollen liver noted in a single low and high dose female or clear fluid contents in the uterus noted in a single high dose female were considered to be related to a physiological variation in pregnancy or to a spontaneous/incidental pathology. No further remarkable differences were noted at post mortem examination in treated animals, when compared with controls, either in animals that completed the treatment or recovery period.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups
Observations of treated animals at removal from the cage and in an open arena did not reveal changes attributable to the test item.
Motor activity, and sensory reaction to stimuli did not show relevant differences between control and treated groups in both sexes. A statistically significant increase in grip strength (second trial) was noted in males of the high dose group (+32%), while the first trial and the relative mean data did not show any differences.

Recovery groups
Observations of treated animals at removal from the cage and in an open arena did not reveal changes attributable to the test item.
No significant alterations in motor activity, grip strength or sensory reaction to stimuli were observed in any treatment group at the examination performed at the end of treatment. A statistically significant decrease in mean grip strength (-37%) was noted in males of the high dose group at the end of treatment. However, was considered of no toxicological significance, since it was not consistent with those observed in the main groups.
At the end of the recovery period, no variations in motor activity, grip strength or sensory reaction to stimuli were observed.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The only relevant change observed in five high dose males randomly selected treated at 10000 ppm when compared with control male animals, consisted in the increased hyaline droplet accumulation in the kidneys. This effect is considered male rat specific and not relevant for humans.
Sporadic lesions, reported in control and treated animals in main and recovery phases, including a malignant nephroblastoma in the kidneys of a single control female of the recovery group and lumen dilatation of uterus in a high dose treated female of the recovery group, were considered to be an expression of spontaneous/or incidental pathology or physiological changes, seen in this species and age in this kind of studies in our experimental conditions.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

In treated females statistically significant decrease was seen in absolute ovaries weight of the high dose group which could be related to the reduction in the number of corpora lutea noted in the animals.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 085 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Remarks on result:

other: The hyaline droplet accumulation in the kidneys of male rats treated at this dose level is considered male rat specific and not relevant for humans.

2

Key result

Dose descriptor:

NOAEL

Effect level:

> 767 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 085 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of the present study, the NOAEL for repeated dose toxicity was considered to be higher than the nominal dose level of 10000 ppm (achieved dosage of 1085 mg/kg bw/day for males and 767 mg/kg bw/day for females).

Executive summary:

The toxic effects on Sprague Dawley rats after oral repeated dosing (via the diet) with Eucalyptus citriodora (Essential oil), was investigated according to OECD TG 422, under GLP. The experiment was performed with 10 rats per dose per sex, including a 2 week recovery. The tested dietary doses corresponded to 1000 ppm, 3000 ppm and 10000 ppm. Males were fed with medicated diet for 2 weeks prior to pairing, during pairing with females until necropsy, for a total of 33 days. Animals of the recovery groups were treated for a total of 4 consecutive weeks and sacrificed after 2 weeks of recovery (Groups 5, 6). Females were fed with medicated diet for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post-partum, for at least 41 days.

 

The following parameters were evaluated: mortality, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), body weight, food consumption, oestrous cycle, clinical pathology investigations (haematology and clinical chemistry), litter data, macroscopic observations, organ weights and histopathological examination. The identification of the stages of the spermatogenic cycle was performed in five randomly selected males of the control and high dose groups.

No mortality occurred throughout the study. No relevant clinical signs, neurotoxicity, clinical pathology or differences in body weight were observed throughout the study in all treated animals of both sexes. Body weight gain decreased in males and females with a dose-related trend. Food consumption was moderately reduced. No toxicological relevance was attributed to the slight reduction noted in terminal body weight of treated males and females. The major changes observed in treated males were an increase in kidney weights in mid- and high dose groups. These changes in kidney weights could be related to male rat specific nephropathic phenomenon which leads to hyaline droplet accumulation. In treated females statistically significant decrease was seen in absolute ovaries weight of the high dose group, likely related to the reduction in the number of corpora lutea noted in the animals. At macroscopic observations, no treatment-related changes were noted.

 

Recovery animals did not show any clinical, or neurological signs or effects on body weight during treatment, or during the recovery phase. Body weight gain and food consumption in treated males and females decreased during the treatment period, but reversed during recovery. During treatment, the food consumption was generally slightly reduced in both sexes especially in the first days of administration. During recovery periods, the food intake become comparable with the control group or, for males, the food intake was higher than control.

At the end of week 2 of the recovery period, changes observed during the dosing phase in haematological and coagulation parameters were no longer observed. After 2 weeks of recovery period, no relevant changes compared to the control group were seen in terminal body weight or organ weights of treated animals. Macroscopically, after 2 weeks of recovery period, no remarkable differences from the control group were noted in previously treated animals.

 

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be higher than the nominal dose level of 10000 ppm (achieved dosage of 1085 mg/kg bw/day for males and 767 mg/kg bw/day for females).