Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-04-2017 to 31-07-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

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:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Light yellow liquid
Details on test material:
Name of test material as cited in study report: Ylang Ylang I
Test Facility Study No. 516037

From start treatment to 24 May 2017 test substance 207779/A was used:
Identification: Ylang Ylang I
Appearance: Light yellow liquid
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Purity/Composition: See Certificate of Analysis
Test substance storage: At room temperature
Stable under storage conditions until: 03 August 2018 (expiry date)

From 24 May 2017 test substance 207779/B was used
Identification: Ylang Ylang I
Appearance: Light yellow liquid
Purity/Composition: See Certificate of Analysis
Test substance storage: At room temperature
Stable under storage conditions until: 18 April 2019 (expiry date) (taken from label)
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 03-08-2018 (batch 1) & 18-04-2019 (batch 2).
- Purity test date: 10-08-2016 (batch 1).

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room tempertature.
- Stability under test conditions: Stability for at least 4 days at room temperature and 2 weeks in freezer is confirmed over the concentration range 500 ppm to 15000 ppm, test facility study no.516039.

FORM AS APPLIED IN THE TEST (if different from that of starting material):
-The test item was mixed without use of a vehicle, directly with the required amount of powder feed.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han
Details on species / strain selection:
Crl: WI(Han) rats were chosen as animal model for this study as it is an accepted rhodent species for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany (female Crl: WI(Han) rats received on 05 Apr 2017, males on 19 Apr 2017).
- Females nulliparous and non-pregnant: yes .
- Age at study initiation: females 13 weeks, males 10 weeks.
- Weight at study initiation: females 204-244 , males 245-289 grams.
- Fasting period before study: no.
- Housing: Macrolon cages, height 18 cm. On arrival and following the pretest (females only) and pre-mating period, 5/cage, MIV type. Mating phase: males and females were cohabitated on a 1:1 basis (MIII type cages). Post-mating phase, males in home cage max 5 males/cage. Females individually housed in MIII type cages. During lactation: females were individually housed in MIII type cage. Bedding: Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany.
- Diet (e.g. ad libitum): Ad libitum standard powder rodent diet during acclimatisation period, ad libitum prepared diet thereafter (except for 2 hours during motor activity measurements).
- Water (e.g. ad libitum): Ad libitum municipal water (except for 2 hours during motor activity measurements).
- Acclimation period: At least 5 days prior to start of the pretest period (females) or at least 5 days before the commencement of administration (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 49-71
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: Not specified.

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The oral route of exposure via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): At least every 14 days.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were prepared and were kept in the freezer (≤-15°C), if not used on the day of preparation. Diets containing test item were stored at room temperature for a maximum of 4 days. Control diet was stored at room temperature for a maximum of 18 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were tested for concentration, homogeneity and stability. Analyses were performed by GC-FID using a validated analytical procedure (test facility study no. 516039).
- Concentration (all groups) results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration.
- Homogeneity (groups 2 and 4) results were considered acceptable if the relative standard deviation (RSD) of concentrations was <= 10%.
- Stability analyses demonstrated that the test item is stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males: treated for 28 days (including mating period)
Females (including 14 days prior to mating):
- With offspring: 49-63 days
- Without offspring: 41-51 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Intended dose level: 0 mg/kg bw
Dose / conc.:
2 500 ppm
Remarks:
Intended dose level: 167 mg/kg bw
Dose / conc.:
7 500 ppm
Remarks:
Intended dose level: 500 mg/kg bw
Dose / conc.:
15 000 ppm
Remarks:
Intended dose level: 1000 mg/kg bw
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the dose levels were selected based on the results of a 14-day dose range finder with dietary administration of Ylang Ylang I in rats (test facility study no. 516038 and in an attempt to produce graded responses to the test item.
- Justification of route: the oral route of administration via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
- Arena observations were perfomed weekly.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: on first day of administration, and twic weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of 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, Days 1, 4, 8, 11, 15, 18, 22, 25 and 29, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and daily during lactation.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified .

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: Not specified .

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: collected on day of sceduled necropsy.
- Anaesthetic used for blood collection: yes, isoflurane.
- Animals fasted: yes, overnight.
- How many animals: selected males and females (5/group).
- Parameters checked: White blood cells (WBC), Neutrophil (absolute), Lymphocyte (absolute), Monocyte (absolute), Eosinophil (absolute), Basophil (absolute), Red blood cells, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: collected on day of sceduled necropsy.
- Animals fasted: yes, overnight.
- How many animals: selected males and females (5/group).
- Parameters checked: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Total Bilirubin, Bile Acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate and Thyroid hormone.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males were examined during week 4, females were examined during the last week of lactation.
- Dose groups that were examined: selected males and females (5/group).
- Battery of functions tested: Hearing ability, Pupillary reflex, Static righting reflex, Fore- and hind-limb grip strength, Locomotor activity.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, post mortem examination for all animals with special attention to the reproductive organs.
-Organs Weighed at Necropsy for selected animals (5/group): Brain; Epididymis; Gland, adrenal; Gland, coagulation; Gland, parathyroid; Gland, prostate; Gland, seminal vesicle; Gland, thyroid; Heart; Kidney; Liver; Ovaries; Spleen; Testes; Thymus; Uterus.
-Organs Weighed at Necropsy for all remaining animals: Epididymis; Gland, coagulation; Gland, parathyroid; Gland, prostate; Gland, seminal vesicle; Gland, thyroid; Testes.

HISTOPATHOLOGY: Yes, basic tissues were collected and preserved for all animals.
-Tissue Collection and Preservation for selected animals (5/group): Animal identification; Artery, aorta; Body cavity, nasopharynx; Bone marrow; Bone, femur; Bone, sternum; Brain (seven levels); Cervix; Epididymis; Esophagus; Eye; Gland, adrenal; Gland, coagulation; Gland, harderian; Gland, lacrimal; Gland, mammary; Gland, parathyroid; Gland, pituitary; Gland, prostate; Gland, salivary; Gland, seminal vesicle; Gland, thyroid; Gross lesions/masses; Gut-associated lymphoid tissue; Heart; Kidney; Large intestine, cecum; Large intestine, colon; Large intestine, rectum; Larynx; Liver; Lung; Lymph node (mandibular and mesenteric site); Muscle, skeletal; Nerve, optic; Nerve, sciatic; Ovaries; Pancreas; Skin; Small intestine, duodenum; Small intestine, ileum; Small intestine, jejunum; Spinal cord; Spleen; Stomach; Testes; Thymus; Tongue; Trachea; Urinary bladder; Uterus; Vagina.
-Tissue Collection and Preservation for all remaining animals (incl. females that failed to deliver pups; nonpregnant and female with total litter loss): Animal identification; Cervix; Epididymis; Gland, coagulation; Gland, mammary; Gland, parathyroid; Gland, pituitary; Gland, prostate; Gland, seminal vesicle; Gland, thyroid; Gross lesions/masses; Ovaries; Testes; Uterus; Vagina.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion.
- Parametric: datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
- Incidence: an overall Fisher’s exact test was used to compare all groups at the 5% significance level.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was noted on a single occasion at the end of lactation for one control female (no. 50), three 7500 ppm females (nos. 62, 68 and 69) and four 15000 ppm females (nos. 73, 76, 79 and 80). For one 15000 ppm female (no. 78) piloerection was noted on three consecutive days during the first week of lactation. These findings where considered incidental and therefore not treatment related.
Mortality:
no mortality observed
Description (incidence):
Female no. 70 (7500 ppm) was euthanized on PND 4 due to total litter loss.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes in body weight or body weight gain were observed in males up to 7500 ppm and in females at 2500 ppm.
In males, body weight and body weight gain was significantly decreased for males at 15000 ppm from Day 4 of the pre-mating phase to Day 15 of the mating period. In females, the following effects were observed:
- At 15000 ppm showed a body weight loss Days 1-11 of the pre mating period. At 15000 ppm differences in body weight gain of -6% for males on Day 15 of the mating period and -5% for females on Day 1 of the mating period were noted, compared to controls.
- In the post-coitum phase, body weights for females treated with 7500 ppm and 15000 ppm were lower compared to controls from Day 0 onwards, which was statistically significant on Day 17 (-7%) for 7500 ppm and 15000 ppm and on Day 20 (-10 %) for 7500 ppm only.
- During lactation, bodyweights for females treated with 7500 ppm and 15000 ppm were lower compared to controls. This was statistically significant for females treated with 15000 ppm during the whole lactation period. For females treated with 7500 ppm this was statistically significant on Day 4 of lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No treatment-related or toxicologically relevant changes in food consumption before or after allowance for body weight were observed in males up to 7500 ppm and for females at 2500 ppm.
In males, food consumption was reduced for 15000 ppm males compared to controls on Days 1-8 of the premating period (both absolute and relative) and Days 1-15 of the mating period. Food consumption corrected for body weight was reduced for 7500 ppm and 15000 ppm males compared to control on Days 1-8 of the mating period. In females of the 15000 ppm dose group, food consumption (both absolute and relative) was reduced compared to controls on Days 1-8 of the premating period, Days 0-20 during the post coitum period and Days 1-5 and Days 6-14 of lactation. For 7500 ppm females food consumption (both absolute and relative) was significantly reduced on Days 1-14 during lactation, which was statistically significant on several days.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males, a statistically significant increase (1.2 fold) in creatinine for males at 15000 ppm compared to control was noted. These findings are considered to be adverse.
In females, an increase in ALP was noted, which was statistically significant at 15000 ppm (3.8-fold increase). Although female no. 76 had a markedly higher ALP value, all other females at 15000 ppm had a higher ALP value compared to controls as well. Other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item related changes included a dose dependent increase in liver weights (relative to body weight was significant), at all dose levels in males and in females at 15000 ppm, in the absence of morphological alterations. This increase in liver weights (>20% compared to control) were considered adverse in males and females at 15000 ppm. In males, a test item-related increase in kidney weights was noted at all doses, but this was considered to be related to alpha-hydrocarbon nephropathy which is not relevant for human. In females, a test item-related increase in kidney weights was noted at 15000 ppm.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In males, a test item-related microscopic findings were noted in the kidney of the 7500 and 15000 ppm treated males (hyaline droplet accumulation increased basophilia and granular casts). The hyaline droplet accumulation was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This is a male rat specific protein and therefore the finding is not considered relevant to humans.
In females, test item-related microscopic findings were noted in the thymus at 15000 ppm (minimal lymphoid depletion).
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
718 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
953 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
718 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 301 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
yes

Any other information on results incl. tables

Test article intake

Group no   2 3 4
Nominal dietary inclusion level (ppm)
2500 7500 15000
Intended dose level (mg/kg bw/day)   167 500 1000
Males
Test article intake (mg/kg bw/day)
Pre-mating 219 (204 - 244) 650 (611 - 687) 1175 (995 - 1127 )
post-mating 257 (190 – 432) 785 (616 - 1100 ) 1426 (1094 - 2192)
Mean of means* 238 718 1301
Females
Test article intake (mg/kg bw/day)
Pre-mating 221 (208 - 245) 651 (581 - 754) 1009 (728 - 1166)
Post-coitum 281 (239 - 316) 832 (710 - 945) 1223 (1130 - 1370)
Lactation 540 (295 - 803) 1449 (667 - 2194) 2738 (1525 - 3614 )
Mean of means* 337 953 1590

* Mean of means of all periods, weighed for number of measurement intervals per period:

Males: ((4 x mean premating) + (4x mean mating)) / 8

Females: ((15 x mean premating) + (20 x mean post-coitum) + (14 x mean lactation)) / 49

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the NOAEL for repeated dose toxicity was established to be 7500 ppm in males and females, corresponding to 718 mg/kg bw/day and 953 mg/kg bw/day respectively. Based on this result, Ylang Ylang I does not need to be classified for specific target organ toxicity following repeated exposure in accordance with the criteria and guidance values outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The repeated dose toxicity of Ylang Ylang I was tested under GLP in a combined repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD TG 422. The experiment was performed by oral administration of the test substance via diet with 10 rats per dose per sex. The tested dietary doses corresponded to 0, 2500, 7500 and 15000 ppm. Males were treated for 28 days (a minimum of two weeks prior to mating and during the mating period). Females that delivered offspring were treated 49-63 days, a minimum of two weeks prior to mating. Females that delivered no healthy offspring were treated for 41-51 days.

The following parameters and endpoints were evaluated in this study for repeated dose toxicology: mortality and moribundity, clinical signs, functional observations and locomotor activity, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

No mortality occurred throughout the study. One female was euthanized on PND 4 due to total litter loss. No relevant clinical signs or neurotoxicity were observed. The treatment related decrease of body weight gain at 15000 ppm for both males (-6%) and females (-10%) were considered non adverse. For females the reduced food consumption at 15000 ppm was considered non adverse. Non-adverse test item related morphological alterations were present in the thymus of females treated at 15000 ppm and consisted of minimal lymphoid depletion. Test item related changes included a dose dependent increase in liver weights (relative to body weight was significant), at all dose levels in males and in females at 15000 ppm, in the absence of morphological alterations.  For females, but not males, an increase in ALP was noted which was statistically significant at 15000 ppm (3.8–fold increase) which in the presence of the increased liver weight was considered adverse. This increase in liver weights (>20% compared to control) were considered adverse in males and females at 15000 ppm. Test item related increase in kidney weights (only relative to body weight was significant) were observed in males and females treated at 15000 ppm. For females the increased kidney weight in the absence of morphological changes was considered non adverse. In males morphological alterations consisted of a combination of increased hyaline droplet accumulation, increased basophilia and granular casts in males treated at 7500 ppm and 15000 ppm. In addition, creatinine levels were statistically significantly increased for males treated with 15000 ppm. The hyaline droplet accumulation was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male is a rat specific protein and is not considered relevant to humans risk assessment.

Under the conditions of this study, the NOAEL for repeated dose toxicity was established to be 7500 ppm in males and females, corresponding to 718 mg/kg bw/day and 953 mg/kg bw/day respectively. Based on this result, Ylang Ylang I does not need to be classified for specific target organ toxicity following repeated exposure in accordance with the criteria and guidance values outlined in Annex I of the CLP Regulation (1272/2008/EC).