Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19-12-2016 to 23-02-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature, protected from light
- Expiration date of the lot/batch: 31 May 2018
- Purity test date: 9 Aug 2016

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 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:201-247 g for males and 192-208 g for females
- Housing:
From arrival to pairing: 5 animals per sex per cage, polysulfone solid bottomed cages. Nesting material was provided and changed at least twice a week.
During mating: 1 male/female pair in clear polysulfone cages with a stainless steel mesh lid and floor. Absorbent material was provided and changed daily.
After mating: Males returned to pre-pairing housing. Females were transferred to individual solid bottomed cages for the gestation period, birth and lactation. Nesting material was provided and changed at least twice 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: 22 December 2016
To: 23 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 (nominal doses: 100, 300 and 1000 mg/kg bw/day). 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 was verified from 1000 to 10000 ppm by chemical analysis (concentration and homogeneity) during the pre-treatment, and during the diet preparation of the main study (first and last week of treatment) period. Stability after 28 hours at room temperature was also verified. Results of all analyses were within the acceptability limits (80-120% for concentration and CV<10% for homogeneity).
Duration of treatment / exposure:
Males: 2 consecutive weeks before pairing, during pairing and followed by 2 consecutive weeks 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 / 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.:
1 000 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.
- 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

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. Animals were checked for mortality early in the day and in the afternoon.

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 (if feeding study):
- 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/group 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 - Pup weight and observation
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

All females were examined for the following:
External and internal abnormalities;

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. The criterion for statistical significance was p<0.05.
Continuous variables: Dunnett’s test or a modified t test, depending on the homogeneity of data.
Histopathological findings: non-parametric Kolmogorov-Smirnov test if n was more than 5.
Other parameters: non-parametric Kruskal-Wallis analysis of variance
Intergroup differences between the control and treated groups: non-parametric version of the Williams test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female animal and its litter of the low dose group were sacrificed on Day 1 post partum because of prolapse of the left horn of the uterus.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption:
- Males (all groups): comparable with the control group.
- Females (high dose 1000 mg/kg bw/day nominal): moderately reduced in the first days of dosing and during pre-mating and gestation.
- Females (medium and low dose 100 and 300 mg/kg bw/day nominal): slight decrease
- During the post partum period, food consumption was slightly reduced in all treated groups.
- During the recovery period, the fluctuations in food intake were considered normal and comparable with the control group.

The mean achieved dosages for the dosing period were found to be slightly lower than theoretical dose levels of 1000, 3000 and 10000 ppm. This discrepancy was due to a lower than expected food consumption. The achieved dosages in mg/kg bw/day, for each group, are presented in the attached tables.
Food efficiency:
not specified
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Main study
Statistically significant fluctuations in some biochemical parameters were recorded generally in all treated groups of males and in the high dose group of females. Due to the low severity and/or the absence of dose-relation, the below findings were considered of no toxicological significance, however additional investigations were performed in the recovery phase.

Males
- decrease of potassium in males dosed at 1000 and 3000 ppm (9% and 8%, respectively)
- decrease of creatinine in males dosed at 3000 ppm (25%)
- increase of globulin in males receiving 3000 and 10000 ppm (10% and 15%, respectively)
- increase of phosphorus in those treated at 10000 ppm (23%)

Females
- triglycerides were increased in 2 females dosed at 3000 ppm and one female receiving 10000 ppm. (3.0 to 4.1 fold)
- decrease of aspartate aminotransferase in females dosed at 10000 ppm (39%)
- decrease of creatinine in females dosed at 10000 ppm (13%)
- increase in bile acids in 2 females treated at 10000 ppm (2.2 and 3.7 fold)

Recovery phase
Creatinine was lower than controls in treated males (23%), these changes were considered to be of no toxicological importance, as values were higher when compared to the dosing phase. The other statistically significant differences between control and treated animals (urea and potassium in males) were not observed during the dosing phase, therefore they were considered unrelated to treatment.
Urinalysis findings:
no effects observed
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):
A slight reduction noted in terminal body weight of high dose male and female animals was not considered toxicologically relevant due to the very slight severity (males: -7%; females: -13%) even if statistically significant for females. Some statistically significant differences were noted in the absolute and/or relative organ weights, such as:
- Reduction in absolute heart weight (-12%) and spleen weight (-18%) in females of the high dose group.
- Increase in relative kidneys weight (+16%) in males of the high dose group.

The variation of the heart weight was probably related to the reduced body weight. The other alterations were not accompanied by histopathological findings and were therefore considered unrelated to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross lesions such as a reduced size of thymus was noted in control and high dose females, a sub-cutaneous pale firm mass was noted in a single low dose female, two pale firm areas in the liver (single lobe) were noted in a single high dose female. Enlarged ovaries or clear fluid in the uterus were considered to be related to a physiological variation in pregnancy or to a spontaneous/incidentalpathology.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Sporadic lesions were reported in control and treated animals in main and recovery phase. These effects 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 and in our experimental conditions.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
An adenocarcinoma was found in the mammary gland of a single treated female. This was considered to be an expression of spontaneous/or incidental pathology and non treatment related.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
708 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Highest dose tested
Key result
Dose descriptor:
NOAEL
Effect level:
771 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:
no

Any other information on results incl. tables

Achieved dosages in the main groups

  Group

  Nominal dosage (mg/kg bw/day)

 ppm

  Achieved dosage (mg/kg bw/day) Mean

  Study achieved dosage (mg/kg bw/day)

 

 

 

  Pre-mating period Males   

  Pre-mating Females

  Mating period Males

Post coitum

Females

Post partum

Females

 Males

  Females

 2

 100

 1000

 75

 84

 71

 84

 107

 73

 88

 3

 300

 3000

 223

 238

 209

 241

 295

 216

 249

 4

 1000

 10000

 718

 704

 699

 769

 909

 708

 771

Achieved dosages in the recovery group

Group

  Nominal dosage (mg/kg bw/day)

 ppm

  Achieved dosage (mg/kg bw/day) Mean

   Achieved dosage (Week 1-2) (mg/kg/day)

 

 

 

  

Days 1-8

Males     

  

Days 1-8

Females

   

Days 8-15

Males

 

Days 8-15

Females

 Males

 

  Females

6

 1000

 10000

 603

 636

 635

 702

 619

 669

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the NOAEL for systemic toxicity was considered to be >708 mg/kg bw/day for males and >771 mg/kg bw/day for females (achieved dosages).
Executive summary:

The toxic effects on Sprague Dawley rats after oral repeated dosing (feeding study) with Citronella oil (Cymbopogon winterianus), 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 period. The tested dietary doses corresponded to 1000 ppm, 3000 ppm and 10000 ppm (nominal doses: 100, 300 and 1000 mg/kg bw/day). Males were fed with medicated diet for 2 weeks prior to pairing, during pairing with females and until necropsy, for a total of 28 days. Animals of the recovery groups were treated for a total of 4 consecutive weeks and sacrificed after 2 weeks of recovery. 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.

During the in vivo phase of the study, no relevant clinical signs, neurotoxic or behavioural signs were observed in any treated main animals of both sexes. Body weight as well as body weight gain of treated males did not show differences of toxicological significance throughout the study. Body weight of females did not show differences of toxicological significance (prior to pairing). The food consumption, in male animals of all groups, was generally comparable with the control group. The food consumption, in female animals was moderately reduced in the first days of dosing in the high dose group while a slight decrease was noted in the other two treatment groups. No adverse findings were recorded in clinical pathology investigations. No toxicological relevance was attributed to an observed slight reduction noted in terminal body weight of treated males and females. The variations in organ weights noted were considered unrelated to treatment since they were not associated with histopathological findings. At macroscopic and microscopic observations, no treatment-related changes were noted. Recovery animals did not show any clinical signs, behavioural/neurological changes, microscopical/macroscopical effects, or changes in terminal body/organ weights. During treatment, the food consumption was slightly reduced in males, while it was moderately reduced in females during the first five days of treatment and generally slightly reduced during the remaining treatment period. During the recovery period, the fluctuations in food intake were considered normal and comparable with the control group.

Based on the results of the present study, the NOAEL for systemic toxicity was considered to be >708 mg/kg bw/day for males and >771 mg/kg bw/day for females (achieved dosages)