Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
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
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
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
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

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
Dose / conc.:
0 ppm
Remarks:
Included in both main and recovery group
Dose / conc.:
1 000 ppm
Remarks:
Intended dose level: 100 mg/kg bw/day
Dose / conc.:
3 000 ppm
Remarks:
Intended dose level: 300 mg/kg bw/day
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
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.
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.
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.
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
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
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).

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

Constituent 1
Chemical structure
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 femaleswere 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
Vehicle:
unchanged (no vehicle)
Details on 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 10000ppm. 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 lastweek 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
Details on study schedule:
- Age at mating of the mated animals in the study: 9-10 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Included in both main and recovery group
Dose / conc.:
1 000 ppm
Remarks:
Intended dose level: 100 mg/kg bw/day
Dose / conc.:
3 000 ppm
Remarks:
Intended dose level: 300 mg/kg bw/day
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:
None

Examinations

Parental animals: Observations and examinations:
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 and motor activity
Oestrous cyclicity (parental animals):
Vaginal smears and oestrus cycle
Sperm parameters (parental animals):
The identification of the stages of the spermatogenic cycle was performed in five randomly selected males of the control and high dose groups.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: The males were killed after the mating of all females, after a total of 32 or 33 days of dosing.
- Maternal animals: The females with live pups were killed on Day 4 post partum.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated ibelow were prepared for microscopic examination and weighed, respectively.
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
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination):
All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities.
All live pups at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

Statistics:
Standard deviationswere 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 theWilliams test. The criterion for statistical significance was p<0.05.
Reproductive indices:
Oestrous cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were measured in treated and control groups.

Males
Copulatory Index (%) = no. of animals mated*100 / no. of animals paired
Fertility Index (%) = no. of males which induced pregnancy*100 / no. of males paired

Females
Copulatory Index (%) = no. of animals mated*100 / no. of animals paired
Fertility Index (%) = no. of pregnant females*100 / no. of females paired
Pre coital Interval = Mean number of days between pairing and mating
Offspring viability indices:
Pre-implantation loss
Pre-birth loss
Post-natal loss
Cumulative pup loss

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant treatment-related 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 weightgain 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 body weight 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):
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 d ose 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 statist ically significant reduced when compared to the control group. Recovery During treatment, the food consumption was generally slightly reduced in both sexes especially in t he 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 thrombopla stin 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/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 femaleswere 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 doserelation, 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
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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decrease in number of corpora lutea and implantation sites (-23% for both parameters) were observed in high dose group when compared to control. As a consequence, reduction in total litter size at birth was noted in high dose group when compared to control values.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 085 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOAEL
Effect level:
242 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
767 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
ovary
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Similar clinical signs were recorded in control and treated pups during the lactation period. The main clinical signs noted in control and treated pups were: cold to touch, apparently no food intake (milk), small and pale appearance. Found dead or missing pups were also observed both in control and treated groups. In addition, one pup of a low dose female was sacrificed for humane reason (moribund status) on Day 1 post partum.
Mortality / viability:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings in pups sacrificed at termination were comparable between groups. Deceased pups did not show relevant findings both in control and treated groups.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 767 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
Maternal expoure
Sex:
male/female
Basis for effect level:
other: Highest dose tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
767 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproductive and developmental toxicity was considered to be 3000 ppm for females (achieved dosage of 242 mg/kg/day) and higher than 10000 ppm for males (achieved dosage of 1085 mg/kg/day).
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 in the parental animals: 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.

The litters were evaluated for: litter size, weight, pup weight, sex ratio, mortality, clinical signs and gross pathology during necropsy.

 

No mortality occurred throughout the study in the parental animals. 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 kidneys weights in mid- and high dose groups. These changes in kidneys 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 (10000 ppm), likely related to the reduction in the number of corpora lutea noted in the animals. At macroscopic observations, no treatment-related changes were noted.

 

Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day), copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage) or fertility index. All pregnant females had a comparable length of gestation period and gave birth on Day 22 post coitum (mean value). Decrease in number of corpora lutea and implantation sites were observed in high dose group (10000 ppm) when compared to control. As a consequence, reduction in total litter size at birth was noted in high dose group when compared to control values. In addition, live litter size at birth and on Day 4 postpartum was decreased in high dose group. Sex ratios at birth and on Day 4 post-partum did not show differences between groups, when calculated as the percentage of males. Similar clinical signs were recorded in control and treated pups during the lactation period. Necropsy findings in pups sacrificed at termination were comparable between groups. Deceased pups did not show relevant findings both in control and treated groups.

 

Based on the results of the present study, the NOAEL for reproductive and developmental toxicity was considered to be 3000 ppm for females (achieved dosage of 242 mg/kg/day) and higher than 10000 ppm for males (achieved dosage of 1085 mg/kg/day).