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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 dec 2017 - 03 dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Justification for type of information:
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 dec 2017 - 03 dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Organisation of Economic Co-operation and Development (OECD) Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
No correction factor required
Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males) and 13 weeks (females)
- Weight at study initiation: 275 - 307 g (males) and 198 and 241 g (females)
- Fasting period before study: No
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals weregroup housed (up to 5 animals of the same sex and same dosing group together) in Macrolon cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon cages.
During the post-mating phase, males were housed in Macrolon cages with a maximum of 5 males/cage. Females were individually housed in Macrolon cages.
During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), adlibitum . The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water: tap water, ad libitum. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water was performed.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 42-73
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 12 Dec 2017 to 27 Jul 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily as a solution and dosed within 4 hours after adding the test item to the vehicle protected from light.
The test item was directly mixed with the vehicle to prevent any loss; therefore vehicle was weighed prior to the test item.
Test item dosing formulations were kept at room temperature protected from light until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Corn oil was used as vehicle for the dose range finder, and therefore selected for the main experiment.
Dose volume: 5 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure.
Stability: During the course of this study at one occasion during the treatment phase of Group 1-4, stability of the prepared formulation was determined at 4 hours at room temperature under protection from light. Duplicate sets of each sample (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Homogeneity: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.
Concentration: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions for suspensions of target concentration.

Details on mating procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which one female that had not shown evidence of mating was separated from the male.
Duration of treatment / exposure:
- Males were exposed for 29-30 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
- Females were exposed for 50-56days, 62 days (one of Group 1 and one of Group 2) or 66 days (one of Group 5) i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy.
- Females which failed to deliver (non-pregnant or implantation site only) were treated for 41 or 43 days, and one non-mated female was treated for 52 days.
- Four females were not dosed on one occasion as these females were littering at the time of dosing.
- Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
The dose volume for each animal was based on the most recent body weight measurement.
- The doses were given using a plastic feeding tube.
- The dosing formulations were stirred continuously during dose administration.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 d/w.
Duration of test:
Males: 29-30 days
Females: 50-56days, 62 days (one of Group 1 and one of Group 2) or 66 days (one of Group 5)
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
This group was added to the study because the evaluation of pup body weight at PND 13 and nipple retention were determined for an insufficient number of litters in Group 1-4 or three litters (1000 mg/kg)). This was considered insufficient to provide conclusive evidence of absence of an effect of treatment and therefore Groups 5 (Control) and 6 (1000 mg/kg) were included to complete this evaluation.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose Range Finding Study was performed:
The dose levels were selected based on the results of a 14-day dose range finder with oral gavage administration of Ethyllinalyl Acetate in rats. In the range finder dose levels of 250, 500 and 1000 mg/
kg were tested.
- Treatment-related slight to moderate salivation was seen in animals treated at 1000 mg/kg.
- At 500 and 250 mg/kg slight salivation was observed. A treatment-related change of the liver of males was observed at macroscopic examinations; 1/5, 4/5 and 5/5 males treated at 250, 500 and 1000 mg/kg, respectively, showed an enlarged liver. This correlated with a treatment-related statistically significant increase in relative liver weights of males. After 14 days of exposure, the enzymatic activities were significantly increased for the PROD, BROD and HCG activities at a dose level of 1000 mg/
kg. No significant increase was noted for the EROD activities in any of the tested dose levels.
- For females, higher relative liver weights were noted as well. The mean relative liver weights were 13% and 28% higher compared to controls at 500 and 1000 mg/kg, respectively (statistically significant at 1000 mg/kg only). The enzymatic activities were significantly increased for the EROD, PROD and HCG activities at the dose levels of 500 mg/kg and 1000 mg/kg and for the BROD a ctivities at the dose level of 1000 mg/kg.
The increased liver weights at 1000 mg/kg were considered acceptable to set the high dose at 1000 mg/kg for the main study. Based on the increase in relative liver weight in males at 500 mg/kg a spacing factor of five was used for dose level selection.
Based in these results, dose levels for the main study 40, 200 and 1000 mg/kg were selected.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology. Only females with live offspring were selected.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- During the dosing period, these observations were performed directly after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on days 1, 4, 7 and 13.
- Terminal body weight were recorded the day of necropsy. Males (fasted) and females (non-fasted).

FOOD CONSUMPTION: Yes.
- Weekly, for males and females. Food consumption was not recorded during the mating period.
- Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on days 1, 4, 7 and 13.

FOOD EFFICIENCY: No.

WATER CONSUMPTION: Yes.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

GENERAL REPRODUCTION DATA: Yes.
- From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
- Females were allowed to litter normally and undisturbed while littering.
- Postnatal day (PND) 1 was defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started).
- The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery.
- Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

BIOANALYSIS AND TOXICOKINETIC EVALUATION: Yes
- On Day 10 of treatment, blood samples (~0.4 mL using K2-EDTA tubes) were collected from the jugular vein from 6 animals/sex/group of Groups 1-4. Animals were restrained during blood collection.
- Samples were collected at 0 (before dosing) and at 15 min, 30 min, 1, 2, 4, 8 and 24 hours (after dosing).

HEMATOLOGY: Yes.
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided
- How many animals: 5 animals/sex/group
- Parameters checked were WBC, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), RBC, reticulocytes, RDW, haemoglobin, haematocrit, MCV, MCY, MCHC, platelets; prothrombin time, activated partial thromboplastin time.
CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.
- Measurement of total ThyroxineT4 was conducted for F0-males (Group 1-4 males only).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes.
Groups 1-4 only
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and
the selected females were tested during the last week of lactation (all before blood sampling).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Sensory activity (Hearing ability, pupillary reflex, static righting reflex),
grip strength (fore and hind limb grip strength), and locomotor activity (total movements and
ambulations).

IMMUNOLOGY: No
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
SACRIFICE
- All culled pups (PND 14-16), except for the two pups per litter selected for blood collection, were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
- The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.
GROSS NECROPSY
- All pups that died or were euthanized were examined externally and sexed (both externally and internally), and their stomachs were examined for the presence of milk. If possible, defects or cause of death were evaluated.
- Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
BLOOD COLLECTION
- From two surplus pups per litter, blood was collected, if possible, and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup.
- In Groups 1-6, blood of F1-animals was collected on PND 4 and PND 14-16, if possible.
- On PND 4, blood was collected from two pups per litter, and the thyroid from two pups per litter was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
-On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Blood was drawn by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.
HISTOPATHOLOGY / ORGAN WEIGTHS
No.
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.
- Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
- Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
- The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1,
Group 4 vs. Group 1, and Group 6 vs. Group 5.
- Parametric: Datasets with at least 2 groups (the designated control group and 1 other group) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: Datasets with at least 2 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. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Indices:
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices
- Post implantation survival index: Total number of offspring born/Total number of uterine implantation
sites x 100
- Live birth index: Number of live offspring on day 1 after littering/Total number of offspring born x100
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x100
- Viability index: Number of live offspring on day 4 before culling/Number live offspring on day 1 after littering x100
- Lactation index: Number of live offspring on Day 13 after littering/Number live offspring on day 4 (after culling) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was noted at 200 and 1000 mg/kg with increasing frequency. At 1000 mg/kg all animals showed salivation. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
From day 4 of treatment onwards an abnormal posture of the tail was noted in all Group 6 animals (1000 mg/kg); for females this was observed up to and including Day 48. Directly after dosing, the rats walked around the cage with their tails held up high and slightly curled. A veterinarian observed this
reaction and it was considered to be most likely related to an unpleasant taste of the formulation. This would also correlate with the observed salivation.
Any other clinical signs noted, including hunched posture and piloerection, occurred incidentally and within the range of background findings. These were identical to those to be expected for rats of this age and strain which are housed and treated under the conditions in this study. In addition, there was no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Female body weights and body weight gains of the 1000 mg/kg groups tended to be higher than the concurrent controls. This was statistically significant for group 6 females during gestation and lactat
ion period. However, the gestation and lactation body weights and body weight gains remained within the historical control ranges and were therefore not considered treatment related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A slight, but statistically significant, increase in absolute and relative food consumption during gestation and lactation period was observed in Group 6, females only. This was not observed in Group 4 females, treated at the same dose level.
Overall, food consumption remained within normal limits and these findings were therefore not considered treatment related.
An isolated statistically significant difference, noted in females (higher relative food consumption at 200 mg/kg between post-coitum days 17-20), was regarded as chance finding, due to the lack of a dose-response relationship.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In Group 4 females (1000 mg/kg), the mean number of platelets was statistically significantly lower compared to controls (relative difference 24%). This was mostly caused by one female, of which the number of platelets was below the P5-value of the historical control range, whereas the other females of the 1000 mg/kg group had normal numbers of platelets (close to the historical control mean).
Therefore, the lower number of platelets of that one female was considered a change finding and not to reflect an effect of the test item.
The statistically significantly lower number of total white blood cells noted in females at 200 mg/kg was regarded as unrelated to treatment due to the lack of a dose-related response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg, the plasma concentrations of chloride (2% lower in males) and cholesterol (31 % higher in females) differed statistically significantly from the concurrent control values.
The mean chloride concentration in 1000 mg/kg males was at the lower end of the historical control range, mean cholesterol in 1000 mg/kg females remained within the normal range.
The statistically significant variations noted in clinical chemistry values at 40 and/or 200 mg/kg were regarded as unrelated to treatment due to the lack of a dose-related response (creatinine and sodium in males, alanine aminotransferase and inorganic phosphate in females).
Thyroid hormone analyses: Serum levels of T4 in males were comparable to concurrent control values.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Treated males had lower mean forelimb and hind limb grip strength values than controls (statistically significant except for hind limb grip strength at 200 mg/kg). The differences showed no dose-related response and grip strength of treated males remained in the normal range. Furthermore, the statistically significant differences could be attributed to the relatively high mean control value. Therefore,these findings were regarded as unrelated to treatment. Grip strength values in females were unremarkable.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
The motor activity in treated animals was comparable to controls. 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):
Liver weights (absolute and relative to body weight) were statistically significantly increased at 200 mg/kg in males and at 1000 mg/kg in both sexes. In males the microscopic correlate for this weight increase was hepatocellular hypertrophy.
Kidney weights (absolute and relative to body weights) were statistically significantly increased at 1000 mg/kg in males. The microscopic correlate for this kidney weight increase was hyaline droplet accumulation.
Any other differences, including those that reached statistical significance (i.e. heart weight and thyroid gland in males, thyroid gland and thymus weights in females) were considered not to be Ethyllinalyl Acetate-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Absolute mean kidney weight of Group 6 females (1000 mg/kg) was statistically significant increased when compared to concurrent control. As the relative kidney weight did not differ significantly from concurrent control, the increase in absolute kidney weight was attributed to the higher body weights of these females at necropsy.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Thymus:
Lymphoid atrophy was noted at a minimal degree in 3/5 females at 1000 mg/kg group.
Thyroid gland:
Mild follicular cell hypertrophy was noted in males at 1000 mg/kg. The minimal follicular cell hypertrophy observed in a few 40 and 200 mg/kg group males was considered to be a background finding.
Liver:
Hepatocellular hypertrophy was noted at a minimal degree in 3/5 males at 1000 mg/kg.
Kidneys:
Hyaline droplet accumulation was noted at increased incidence and severity (5/5 moderate) in males at 1000 mg/kg. A background level of this finding (at minimal to slight degree) was present in 3/5 control males.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was not affected by treatment. The survival indices were 98 and 94%, for control Group 1 and 5, respectively. In the treatment groups, the survival indices were 90, 90, 93 and 88% for Group 2, 3, 4 and 6, respectively.
The post-implantation survival index of Group 6 (1000 mg/kg) was slightly lower when compared to the control groups. As the index of Group 6 remained within the historical control range (period 2015-2018: mean = 92, P5 - P95 = 83 – 99 (n=98)), the apparently lower post-implantation survival index of Group 6 was considered not to be test item-related.
For one female (Group 2, 40 mg/kg) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
Gestation index and duration of gestation were not affected by treatment.
- Except for one control female of Group 5 (control) and one female at Group 2 (40 mg/kg), all pregnant females had live offspring.
- The gestation indices were 90% for Group 5 (control), 89% for the Group 2 (40 mg/kg) and 100% for the other groups.
- These cases of failed pregnancy occurred without related histopathology changes in reproductive organs.
- The failed pregnancy at 40 mg/kg was regarded as unrelated to treatment due to the incidental occurrence and the lack of a dose-related trend.
Details on maternal toxic effects:
PRECOITAL TIME: not affected by treatment.
Most mated females showed evidence of mating within four days.
NUMBER OF IMPLANTATION SITES: not affected by treatment.
One control female and one female of the 40 mg/kg group had only one implantation site (they had no offspring). All other females treated with the test item had normal numbers of implantation sites.
Therefore, the incidental finding in a low-dose female was regarded as unrelated to treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no maternal toxicity was observed
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no developmental toxicity was observed
Fetal body weight changes:
not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
not examined
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.

Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings.
Remarks on result:
not measured/tested
Remarks:
Embryotoxic / teratogenic effects:no effects. Remark: Only early postnatal pup development parameters were examined including body weight, post-natal loss, sex ratio, clinical signs, body weight and external macroscopy.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

REPRODUCTIVE DATA

Length and regularity of the estrous cycle, mating index, precoital time, number of implantation sites and fertility index were not affected by treatment.

 

EARLY POSTNATAL PUP DEVELOPMENT

No effects observed.

 

MORTALITY PUPS

The number of live offspring on PND 1 as percentage of total number of offspring born was not affected by treatment.

At first litter check, four control pups (one litter, Group 5), six pups in the 40 mg/kg group (3 litters, group 2) and two pups in the 1000 mg/kg (two litters, group 6) were found dead. This pup mortality, which remained within normal limits, was judged to be unrelated to treatment due to the lack of a dose-related trend.

 

CLINICAL SIGNS PUPS

The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

One pup at 1000 mg/kg showed a distended abdomen on PND 11-13. This pup was inspected by a veterinarian and based on the presence of milk in the stomach, normal colour and temperature, the pup was not sacrificed in extremis. However, on PND 14 the pup went missing (presumed cannibalized) and therefore no macroscopic correlate could be made to this clinical observation. Based on the incidental occurrence, this clinical sign was considered to be unrelated to treatment.

 

BODY WEIGHT PUPS

No effects observed.

 

MACROSCOPY PUPS

No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of the few findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Conclusions:
In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect levels (NOAELs) of Ethyllinalyl Acetate were established:

Parental NOAEL: at least 1000 mg/ kg, based on the absence of adverse effects. Corresponding Cmax values on Day 10 of treatment were 314 and 1330 ng/mL for males and females, respectively and AUC (last) values were 1560 and 7620 h ng/mL, for males and females respectively.

Reproduction NOAEL: at least 1000 mg/kg, based on the absence of adverse effects.
Developmental NOAEL: at least 1000 mg/kg, based on the absence of adverse effects.
Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. Ethyllinalyl Acetate was administered by daily oral gavage to male and female rats at dose levels of 0, 40, 200 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 50-66 days).

Blood for toxicokinetic evaluation was sampled on treatment Day 10 (from pre-dose up to

24 hours post-dose). Blood was analysed for the concentrations of the test item and a defined metabolite i.e. Ethyllinalool. A toxicokinetic evaluation was performed.

No mortality was observed during the study. Few non-adverse test items-related findings at 200 mg/kg and 1000 mg/kg, such as salivation, lower chloride concentration (males) and higher cholesterol concentration (females). Increased liver weights in males and females were found. The increase of liver weight was considered an adaptive response, due to the induction of liver enzyme activities in both sexes. Increased kidney weight was associated to a hyaline droplet accumulation, most likely representing alpha 2 microglobuline. There was no indication of tubular damage, therefore these effects are considered no adverse. Moreover, this finding is specific for male rats and it is not relevant for humans. Mild effects were also observed in thymus and thyroids and are considered no adverse.

No treatment-related changes were noted in the reproductive parameters examined (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

No treatment-related changes were observed in the developmental parameters examined (i.e. gestation index and duration, parturition/maternal care, post-impantation survival index, litter size, live birth index, viability index, lactation index, clinical signs, body weights, sex ratio of the pups, and anogenital distance, areola/nipple retention. No effect on T4 levels of the pups were found.

Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 dec 2017 - 03 dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)
Version / remarks:
July 2000
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)
- Guideline on Bioanalytical Method Validation, European Medicines Agency (EMA), EMEA/CHMP /EWP/192217/2009, 21 July 2011
- Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM), May 2001
- ICH M3 (R2). Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals. December 2009
- ICH S3a. Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies, October 1994.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
No correction factor required
Species:
rat
Strain:
other: Crl:WI(Han).
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males) and 13 weeks (females)
- Weight at study initiation: 275 - 307 g (males) and 198 and 241 g (females)
- Fasting period before study: No
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in Macrolon cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon cages. During the post-mating phase, males were housed in Macrolon cages with a maximum of 5 males/cage. Females were individually housed in Macrolon cages.
During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), adlibitum . The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water: tap water, ad libitum. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water was performed.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 42-73
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 12 Dec 2017 to 27 Jul 2018
Route of administration:
oral: gavage
Details on exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily as a solution and dosed within 4 hours after adding the test item to the vehicle protected from light.
The test item was directly mixed with the vehicle to prevent any loss; therefore vehicle was weighed prior to the test item.
Test item dosing formulations were kept at room temperature protected from light until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Corn oil was used as vehicle for the dose range finder, and therefore selected for the main experiment.
Dose volume: 5 mL/kg body weight.
Details on mating procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which one female that had not shown evidence of mating was separated from the male.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure.
Stability: During the course of this study at one occasion during the treatment phase of Group 1-4, stability of the prepared formulation was determined at 4 hours at room temperature under protection from light. Duplicate sets of each sample (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Homogeneity: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.
Concentration: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions for suspensions of target concentration.

Duration of treatment / exposure:
- Males were exposed for 29-30 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
- Females were exposed for 50-56days, 62 days (one of Group 1 and one of Group 2) or 66 days (one of Group 5) i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy.
- Females which failed to deliver (non-pregnant or implantation site only) were treated for 41 or 43 days, and one non-mated female was treated for 52 days.
- Four females were not dosed on one occasion as these females were littering at the time of dosing.
Frequency of treatment:
Once daily for 7 d/w.
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 15 weeks.
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
This group was added to the study because the evaluation of pup body weight at PND 13 and nipple retention were determined for an insufficient number of litters in Group 1-4 or three litters (1000 mg/kg)). This was considered insufficient to provide conclusive evidence of absence of an effect of treatment and therefore Groups 5 (Control) and 6 (1000 mg/kg) were included to complete this evaluation.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose Range Finding Study was performed:
The dose levels were selected based on the results of a 14-day dose range finder with oral gavage administration of Ethyllinalyl Acetate in rats. In the range finder dose levels of 250, 500 and 1000 mg/kg were tested.
- Treatment-related slight to moderate salivation was seen in animals treated at 1000 mg/kg.
- At 500 and 250 mg/kg slight salivation was observed. A treatment-related change of the liver of males was observed at macroscopic examinations; 1/5, 4/5 and 5/5 males treated at 250, 500 and 1000
mg/kg, respectively, showed an enlarged liver. This correlated with a treatment-related statistically significant increase in relative liver weights of males. After 14 days of exposure, the enzymatic activities were significantly increased for the PROD, BROD and HCG activities at a dose level of 1000 mg/kg. No significant increase was noted for the EROD activities in any of the tested dose levels.
- For females, higher relative liver weights were noted as well. The mean relative liver weights were 13% and 28% higher compared to controls at 500 and 1000 mg/kg, respectively (statistically significant at 1000 mg/kg only). The enzymatic activities were significantly increased for the EROD, PROD and HCG activities at the dose levels of 500 mg/kg and 1000 mg/kg and for the BROD activities at the dose level of 1000 mg/kg.
The increased liver weights at 1000 mg/kg were considered acceptable to set the high dose at 1000 mg/kg for the main study. Based on the increase in relative liver weight in males at 500 mg/kg a spacing factor of five was used for dose level selection.

Based in these results, dose levels for the main study 40, 200 and 1000 mg/kg were selected.

Selection of animals for selected measurements:
5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology. Only females with live offspring were selected.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- During the dosing period, these observations were performed directly after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on days 1, 4, 7 and 13.
- Terminal body weight were recorded the day of necropsy. Males (fasted) and females (non-fasted).

FOOD CONSUMPTION: Yes.
- Weekly, for males and females. Food consumption was not recorded during the mating period.
- Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on days 1, 4, 7 and 13.

FOOD EFFICIENCY: No.

WATER CONSUMPTION: Yes.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

GENERAL REPRODUCTION DATA: Yes.
- From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.
- Females were allowed to litter normally and undisturbed while littering.
- Postnatal day (PND) 1 was defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started).
- The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery.
- Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.

BIOANALYSIS AND TOXICOKINETIC EVALUATION: Yes
- On Day 10 of treatment, blood samples (~0.4 mL using K2-EDTA tubes) were collected from the jugular vein from 6 animals/sex/group of Groups 1-4. Animals were restrained during blood collection.
- Samples were collected at 0 (before dosing) and at 15 min, 30 min, 1, 2, 4, 8 and 24 hours (after dosing).

HEMATOLOGY: Yes.
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided
- How many animals: 5 animals/sex/group
- Parameters checked were WBC, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), RBC, reticulocytes, RDW, haemoglobin, haematocrit, MCV, MCY, MCHC, platelets; prothrombin time, activated partial thromboplastin time.
CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.
- Measurement of total ThyroxineT4 was conducted for F0-males (Group 1-4 males only).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes.
Groups 1-4 only
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and
the selected females were tested during the last week of lactation (all before blood sampling).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Sensory activity (Hearing ability, pupillary reflex, static righting reflex),
grip strength (fore and hind limb grip strength), and locomotor activity (total movements and
ambulations).

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
Sperm parameters (parental animals):
Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
On PND 1: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, anogenital distance (AGD) .
On PND1 and daily thereafter: mortality, physical or behavioural abnormalities.
On PND 1, 4, 7 and 13: body weights.
On PND 13: presence of nipples/areolae in male pups.

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
Yes, full post-mortem necropsy, with special attention paid to the reproductive organs.
The following organ weights were recorded:
- On selected 5 animals/sex/group: adrenal glands, brain, epididymis, heart, kidneys, liver, ovaries, prostates, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid including parathyroid if detectable, uterus including cervix.
- All remaining animals: epididymis, prostate, seminal vesicles including coagulation glands, testes, thyroid

HISTOPATHOLOGY: Yes
Groups 1 to 4:
- On selected 5 animals/sex/group in control and high-dose groups and all animals that were euthanized in extremis: Aorta, nasopharynx, bone marrow, bones (femur, sternum), brain (seven levels), cervix, epididymis, esophagus, eyes (with optic nerve if available), glands (adrenal, coagulation, harderian, lacrimal, mammary, parathyroid (if present in section of the thyroid), pituitary, prostate, salivary, seminal vesicle, thyroid), gross lesions/masses, gut-associated lymphoid tissue, heart, kidneys, Intestine (duodenum, jejunum, ileum, cecum, colon, rectum), larynx, liver, lungs, lymph nodes (mandibular, mesenteric), sciatic nerve, ovaries, pancreas, skin, spinal cord, spleen, stomach, testes, thymus, tongue, trachea, urinary bladder, uterus, vagina.
- On selected 5 males in all groups and all males that failed to sire: additional testes slides.
- On all animals: all gross lesions.
- All remaining animals, including males that failed to sire and females that failed to deliver pups: Cervix, epididymis, glands (coagulation, mammary, parathyroid, pituitary, prostate, seminal vesicle, and thyroid), gross lesions/masses, ovaries, testes, uterus, vagina.
Groups 5 and 6:
- On all animals: Cervix, epididymis, glands (coagulation, mammary, parathyroid, pituitary, prostate, seminal vesicle, and thyroid), gross lesions/masses, kidneys, liver, ovaries, testes, thymus, uterus, vagina.

- Estrous cycle examinations: Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.
- Thyroid hormone measurements: Serum T4 concentration was measured in males prior to scheduled sacrifice.
Postmortem examinations (offspring):
SACRIFICE
- All culled pups (PND 14-16), except for the two pups per litter selected for blood collection, were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20%).
- The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
- All pups that died or were euthanized were examined externally and sexed (both externally and internally), and their stomachs were examined for the presence of milk. If possible, defects or cause of death were evaluated.
- Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.

BLOOD COLLECTION

- From two surplus pups per litter, blood was collected, if possible, and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much blood as possible was collected from this single pup.
- In Groups 1-6, blood of F1-animals was collected on PND 4 and PND 14-16, if possible.
- On PND 4, blood was collected from two pups per litter, and the thyroid from two pups per litter was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
-On PND 14-16, separate blood samples were collected from two pups per litter (from one male and one female). Blood was drawn by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.
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.
- Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
- Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
- The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1, Group 4 vs. Group 1, and Group 6 vs. Group 5.
- Parametric: Datasets with at least 2 groups (the designated control group and 1 other group) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: Datasets with at least 2 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.
The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall testis significant.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:
- Post implantation survival index: Total number of offspring born/Total number of uterine implantation sites x 100
- Live birth index: Number of live offspring on day 1 after littering/Total number of offspring born x100
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x100
- Viability index: Number of live offspring on day 4 before culling/Number live offspring on day 1 after littering x100
- Lactation index: Number of live offspring on Day 13 after littering/Number live offspring on day 4 (after culling) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was noted at 200 and 1000 mg/kg with increasing frequency. At 1000 mg/kg all animals showed salivation. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
From day 4 of treatment onwards an abnormal posture of the tail was noted in all Group 6 animals (1000 mg/kg); for females this was observed up to and including Day 48. Directly after dosing, the rats walked around the cage with their tails held up high and slightly curled. A veterinarian observed this reaction and it was considered to be most likely related to an unpleasant taste of the formulation. This would also correlate with the observed salivation.
Any other clinical signs noted, including hunched posture and piloerection, occurred incidentally and within the range of background findings. These were identical to those to be expected for rats of this age and strain which are housed and treated under the conditions in this study. In addition, there was no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Female body weights and body weight gains of the 1000 mg/kg groups tended to be higher than the concurrent controls. This was statistically significant for group 6 females during gestation and lactation period. However, the gestation and lactation body weights and body weight gains remained within the historical control ranges and were therefore not considered treatment related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A slight, but statistically significant, increase in absolute and relative food consumption during gestation and lactation period was observed in Group 6, females only. This was not observed in Group 4 females, treated at the same dose level.
Overall, food consumption remained within normal limits and these findings were therefore not considered treatment related.
An isolated statistically significant difference, noted in females (higher relative food consumption at 200 mg/kg between post-coitum days 17-20), was regarded as chance finding, due to the lack of a dose-response relationship.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In Group 4 females (1000 mg/kg), the mean number of platelets was statistically significantly lower compared to controls (relative difference 24%). This was mostly caused by one female, of which the number of platelets was below the P5-value of the historical control range, whereas the other females of the 1000 mg/kg group had normal numbers of platelets (close to the historical control mean).
Therefore, the lower number of platelets of that one female was considered a change finding and not to reflect an effect of the test item.
The statistically significantly lower number of total white blood cells noted in females at 200 mg/kg was regarded as unrelated to treatment due to the lack of a dose-related response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg, the plasma concentrations of chloride (2% lower in males) and cholesterol (31 % higher in females) differed statistically significantly from the concurrent control values.
The mean chloride concentration in 1000 mg/kg males was at the lower end of the historical control range, mean cholesterol in 1000 mg/kg females remained within the normal range.
The statistically significant variations noted in clinical chemistry values at 40 and/or 200 mg/kg were regarded as unrelated to treatment due to the lack of a dose-related response (creatinine and sodium in males, alanine aminotransferase and inorganic phosphate in females).
Thyroid hormone analyses: Serum levels of T4 in males were comparable to concurrent control values.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Treated males had lower mean forelimb and hind limb grip strength values than controls (statistically significant except for hind limb grip strength at 200 mg/kg). The differences showed no dose-related response and grip strength of treated males remained in the normal range. Furthermore, the statistically significant differences could be attributed to the relatively high mean control value. Therefore, these findings were regarded as unrelated to treatment. Grip strength values in females were unremarkable.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
The motor activity in treated animals was comparable to controls. 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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Thymus:
Lymphoid atrophy was noted at a minimal degree in 3/5 females at 1000 mg/kg group.
Thyroid gland:
Mild follicular cell hypertrophy was noted in males at 1000 mg/kg. The minimal follicular cell hypertrophy observed in a few 40 and 200 mg/kg group males was considered to be a background finding.
Liver:
Hepatocellular hypertrophy was noted at a minimal degree in 3/5 males at 1000 mg/kg.
Kidneys:
Hyaline droplet accumulation was noted at increased incidence and severity (5/5 moderate) in males at 1000 mg/kg. A background level of this finding (at minimal to slight degree) was present in 3/5 control males.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
One female of the 1000 mg/kg group was acyclic. That female had a normal precoital interval (evidence of mating after four days of cohabitation) and a normal litter. Moreover, there were no treatment-related changes in the morphology of the female reproductive tract. Therefore, the acyclicity of one female was considered a change finding and not to reflect an effect of the test item on estrous cyclicity.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Mating index was not affected by treatment.
The unsuccessful mating of one couple of the lowest dose group, without supportive morphological changes in reproductive organs, was judged to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.
Fertility index was considered not to be affected by treatment.
Except for two females treated at 1000 mg/kg (one in Group 4 and one in Group 6), all mated females were pregnant. The fertility indices were 90% for both the 1000 mg/kg groups and 100% for the other groups. The two cases of non-pregnancy were within the range to be expected for this strain of rats and type of study, see also the historical control range. In addition, no related histopathology changes were noted in reproductive organs. Therefore, the non-pregnancies in the 1000 mg/kg groups were regarded as unrelated to treatment.
MATING INDEX: not affected by treatment.
The unsuccessful mating of one couple of the lowest dose group, without supportive morphological changes in reproductive organs, was judged to be unrelated to treatment due to the incidental occurrence and lack of a dose-related trend.

PRECOITAL TIME: not affected by treatment.
Most mated females showed evidence of mating within four days.

NUMBER OF IMPLANTATION SITES: not affected by treatment.
One control female and one female of the 40 mg/kg group had only one implantation site (they had no offspring). All other females treated with the test item had normal numbers of implantation sites. Therefore, the incidental finding in a low-dose female was regarded as unrelated to treatment.

FERTILITY INDEX: not affected by treatment.
Except for two females treated at 1000 mg/kg (one in Group 4 and one in Group 6), all mated females were pregnant. The fertility indices were 90% for both the 1000 mg/kg groups and 100% for the other groups.
The two cases of non-pregnancy were within the range to be expected for this strain of rats and type of study. Moreover, no related histopathology changes were noted in reproductive organs. Therefore, the two non-pregnancies in the 1000 mg/kg groups were regarded as unrelated to treatment.

GESTATION INDEX AND DURATION: not affected by treatment.
Except for one control female of Group 5 and one female at 40 mg/kg, all pregnant females had live offspring. The gestation indices were 90% for Group 5, 89% for the 40 mg/kg group and 100% for the other groups.
These cases of failed pregnancy occurred without related histopathology changes in reproductive organs. The failed pregnancy at 40 mg/kg was regarded as unrelated to treatment due to the incidental occurrence and the lack of a dose-related trend.

PARTURITION/MATERNAL CARE: not affected by treatment.
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
No deficiencies in maternal care were observed.

POSTIMPLANTATION INDEX: not affected by treatment.
The survival indices were 98 and 94%, for control Group 1 and 5, respectively. In the treatment groups, the survival indices were 90, 90, 93 and 88% for Group 2, 3, 4 and 6, respectively.
The post-implantation survival index of Group 6 (1000 mg/kg) was slightly lower when compared to the control groups. As the index of Group 6 remained within the historical control range ((period 2015-2018): mean = 92, P5 - P95 = 83 – 99 (n=98)), the apparently lower post-implantation survival index of Group 6 was considered not to be test item-related.
For one female (at 40 mg/kg) the number of pups born was slightly higher than the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.

LITTER SIZE: not affected by treatment.



Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed up to the highest dose level tested for systemic toxicity as well as fertility. .
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
One pup at 1000 mg/kg showed a distended abdomen on PND 11-13. This pup was inspected by a veterinarian and based on the presence of milk in the stomach, normal colour and temperature, the pup was not sacrificed in extremis. However, on PND 14 the pup went missing (presumed cannibalized) and therefore no macroscopic correlate could be made to this clinical observation. Based on the incidental occurrence, this clinical sign was considered to be unrelated to treatment.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on PND 1 as percentage of total number of offspring born was not affected by treatment.
At first litter check, four control pups, six pups in the 40 mg/kg group and two pups in the 1000 mg/kg were found dead. This pup mortality, which remained within normal limits, was judged to be unrelated to treatment due to the lack of a dose-related trend.

- Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 98 and 99%, for control Group 1 and 5, respectively. In the treatment groups, the survival indices were 99, 98, 97 and 99% for Group 2, 3, 4 and 6, respectively. Four pups of the control groups, one pup at 40 mg/kg, two pups at 200 mg/kg and five pups at 1000 mg/kg went missing, presumed cannibalized or were found dead/euthanized in extremis between PND 1-4. This pup mortality was regarded as unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.
- Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. All groups had a lactation index of 100%. At PND 14, one pup at 1000 mg/kg went missing. As this pup was still alive on PND 13, this did not affect the lactation index
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:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were not affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio was not affected by treatment.

Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the few findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed in F1 generation.
Key result
Reproductive effects observed:
no
Treatment related:
no

Analysis of dose preparations:

Accuracy: The concentrations analyzed in the formulations of Groups 2, 3, 4 and 6 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

No test item was detected in the Group 1 and 5 formulations.

Homogeneity: The formulations of Groups 2, 4 and 6 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability: Formulations of Groups 2 and 4 were stable when stored at room temperature protected from light for at least four hours (i.e. relative difference over the storage period ≤10%).

Conclusions:
In an oral OECD 422 screening study with rats, the NOAEL was determined to be at least 1000 mg/kg bw/day, based on no observed adverse effects at the highest dose tested. No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).
Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. Ethyllinalyl Acetate was administered by daily oral gavage to male and female rats at dose levels of 0, 40, 200 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 50-66 days).

Blood for toxicokinetic evaluation was sampled on treatment Day 10 (from pre-dose up to

24 hours post-dose). Blood was analysed for the concentrations of the test item and a defined metabolite i.e. Ethyllinalool. A toxicokinetic evaluation was performed.

No mortality was observed during the study. Few non-adverse test items-related findings at 200 mg/kg and 1000 mg/kg, such as salivation, lower chloride concentration (males) and higher cholesterol concentration (females). Increased liver weights in males and females were found. The increase of liver weight was considered an adaptive response, due to the induction of liver enzyme activities in both sexes. Increased kidney weight was associated to a hyaline droplet accumulation, most likely representing alpha 2 microglobuline. There was no indication of tubular damage, therefore these effects are considered no adverse. Moreover, this finding is specific for male rats and it is not relevant for humans. Mild effects were also observed in thymus and thyroids and are considered no adverse.

No treatment-related changes were noted in the reproductive parameters examined (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

 

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect levels (NOAELs) of Ethyllinalyl Acetate were established:

 

Parental NOAEL: at least 1000 mg/ kg, based on the absence of adverse effects. Corresponding Cmax values on Day 10 of treatment were 314 and 1330 ng/mL for males and females, respectively and AUC (last) values were 1560 and 7620 h ng/mL, for males and females respectively.

 

Reproduction NOAEL: at least 1000 mg/kg, based on the absence of adverse effects. 

 

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)
Version / remarks:
July 2000
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)
- Guideline on Bioanalytical Method Validation, European Medicines Agency (EMA), EMEA/CHMP / EWP/192217/2009, 21 July 2011
- Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM), May 2001
- ICH M3 (R2). Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals. December 2009
- ICH S3a. Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies, October 1994.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance: clear liquid
- Storage conditions: at room temperature protected from light, container flushed with nitrogen, desiccated
Specific details on test material used for the study:
No correction factor required

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males) and 13 weeks (females)
- Weight at study initiation: 261 - 295 g (males) and 209 and 241 g (females)
- Fasting period before study: No
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in Macrolon cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon cages.
During the post-mating phase, males were housed in Macrolon cages with a maximum of 5 males/cage. Females were individually housed in Macrolon cages.
During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups
were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), adlibitum . The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water: tap water, ad libitum. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water was performed.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 38-74
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 12 Dec 2017 to 27 Jul 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily as a solution and dosed within 4 hours after adding the test item to the vehicle protected from light.
The test item was directly mixed with the vehicle to prevent any loss; therefore vehicle was weighed prior to the test item.
Test item dosing formulations were kept at room temperature protected from light until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Dose volume: 5 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure.
Stability: During the course of this study at one occasion during the treatment phase of Group 1-4, stability of the prepared formulation was determined at 4 hours at room temperature under protection from light. Duplicate sets of each sample (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Homogeneity: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.
Concentration: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions for suspensions of target concentration.
Duration of treatment / exposure:
- Males were exposed for 29-30 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
- Females were exposed for 50-56days, 62 days (one of Group 1 and one of Group 2) or 66 days (one of Group 5) i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy.
- Females which failed to deliver (non-pregnant or implantation site only) were treated for 41 or 43 days, and one non-mated female was treated for 52 days.
- Four females were not dosed on one occasion as these females were littering at the time of dosing.
Frequency of treatment:
Once daily, 7 d/w
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
This group was added to the study because the evaluation of pup body weight at PND 13 and nipple retention were determined for an insufficient number of litters in Group 1-4 or three litters (1000 mg/kg)). This was considered insufficient to provide conclusive evidence of absence of an effect of treatment and therefore Groups 5 (Control) and 6 (1000 mg/kg) were included to complete this evaluation
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of a 14-day dose range finder with oral gavage administration of Ethyllinalyl Acetate in rats.
In the range finder dose levels of 250, 500 and 1000 mg/kg were tested.
- Treatment-related slight to moderate salivation was seen in animals treated at 1000 mg/kg.
- At 500 and 250 mg/kg slight salivation was observed. A treatment-related change of the liver of males was observed at macroscopic examinations; 1/5, 4/5 and 5/5 males treated at 250, 500 and 1000 mg/kg, respectively, showed an enlarged liver. This correlated with a treatment-related statistically significant increase in relative liver weights of males. After 14 days of exposure, the enzymatic activities were significantly increased for the PROD, BROD and HCG activities at a dose level of 1000 mg/kg. No significant increase was noted for the EROD activities in any of the tested dose levels.
- For females, higher relative liver weights were noted as well. The mean relative liver weights were 13% and 28% higher compared to controls at 500 and 1000 mg/kg, respectively (statistically significant at 1000 mg/kg only). The enzymatic activities were significantly increased for the EROD, PROD and HCG activities at the dose levels of 500 mg/kg and 1000 mg/kg and for the BROD activities at the dose level of 1000 mg/kg.
The increased liver weights at 1000 mg/kg were considered acceptable to set the high dose at 1000 mg/kg for the main study. Based on the increase in relative liver weight in males at 500 mg/kg a spacing factor of five was used for dose level selection.

As dose levels for the main study 40, 200 and 1000 mg/kg were selected.
Positive control:
No.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: Twice daily.

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

BODY WEIGHT:
Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on days 1, 4, 7 and 13
- Terminal body weight were recorded the day of necropsy. Males (fasted) and females (non-fasted).

FOOD CONSUMPTION:
Yes. Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on days 1, 4, 7 and 13.

FOOD EFFICIENCY:
No.

WATER CONSUMPTION: Yes.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

BIOANALYSIS AND TOXICOKINETIC EVALUATION: Yes
- On Day 10 of treatment, blood samples (~0.4 mL using K2-EDTA tubes) were collected from the jugular vein from 6 animals/sex/group of Groups 1-4. Animals were restrained during blood collection.
- Samples were collected at 0 (before dosing) and at 15 min, 30 min, 1, 2, 4, 8 and 24 hours (after dosing).
- Parameters that were calculated included tlast, tmax, Cmax, AUClast, dose effect, sex effect and exposure to metabolite versus parent. The t1/2 value could only be calculated in females at the high dose. For low- and mid-dose females and all groups of males t1/2 values could not be calculated since no log linear regression was possible.

HEMATOLOGY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were WBC, differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), RBC, reticulocytes, RDW, haemoglobin, haematocrit, MCV, MCH, MCHC, platelets; prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: ALAT, ASAT, ALP, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate
- Measurement of total ThyroxineT4 was conducted for F0-males (Group 1-4 males only). For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes (Groups 1-4 only)
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during the last week of lactation (all before blood sampling).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: Sensory activity (Hearing ability, pupillary reflex, static righting reflex), grip strength (fore and hind limb grip strength), and locomotor activity (total movements and ambulations).

IMMUNOLOGY: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Yes, full post-mortem necropsy, with special attention paid to the reproductive organs.
The following organ weights were recorded for groups 1-4:
- On selected 5 animals/sex/group: adrenal glands, brain, epididymis, heart, kidneys, liver, ovaries, prostates, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid including parathyroid if detectable, uterus including cervix.
- All remaining animals: epididymis, prostate, seminal vesicles including coagulation glands, testes, thyroid.
The following organ weights were recorded for groups 5-6:
-epidymis, seminal vesicle, coagulation glands, prostate, testes, thyroid and parathyroid.

HISTOPATHOLOGY: Yes
Groups 1 to 4:
- On selected 5 animals/sex/group in control and high-dose groups and all animals that were euthanized in extremis: Aorta, nasopharynx, bone marrow, bones (femur, sternum), brain (seven levels), cervix, epididymis, esophagus, eyes (with optic nerve if available), glands (adrenal, coagulation, harderian, lacrimal, mammary, parathyroid (if present in section of the thyroid), pituitary, prostate, salivary, seminal vesicle, thyroid), gross lesions/masses, gut-associated lymphoid tissue, heart, kidneys, Intestine (duodenum, jejunum, ileum, cecum, colon, rectum), larynx, liver, lungs, lymph nodes (mandibular, mesenteric), sciatic nerve, ovaries, pancreas, skin, spinal cord, spleen, stomach, testes, thymus, tongue, trachea, urinary bladder, uterus, vagina.
- On selected 5 males in all groups and all males that failed to sire: additional testes slides.
- On all animals: all gross lesions.
- All remaining animals, including males that failed to sire and females that failed to deliver pups: Cervix, epididymis, glands (coagulation, mammary, parathyroid, pituitary, prostate, seminal vesicle, and thyroid), gross lesions/masses, ovaries, testes, uterus, vagina.
Groups 5 and 6:
- On all animals: Cervix, epididymis, glands (coagulation, mammary, parathyroid, pituitary, prostate, seminal vesicle, and thyroid), gross lesions/masses, kidneys, liver, ovaries, testes, thymus, uterus, vagina.
Other examinations:
- Estrous cycle examinations: Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period.

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.
- Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
- Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
- The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1,
Group 4 vs. Group 1, and Group 6 vs. Group 5.
- Parametric: Datasets with at least 2 groups (the designated control group and 1 other group) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: Datasets with at least 2 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. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was noted at 200 and 1000 mg/kg with increasing frequency. At 1000 mg/kg all animals showed salivation. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
From day 4 of treatment onwards an abnormal posture of the tail was noted in all Group 6 animals (1000 mg/kg); for females this was observed up to and including Day 48. Directly after dosing, the rats walked around the cage with their tails held up high and slightly curled. A veterinarian observed this reaction and it was considered to be most likely related to an unpleasant taste of the formulation. This would also correlate with the observed salivation.
Any other clinical signs noted, including hunched posture and piloerection, occurred incidentally and within the range of background findings. These were identical to those to be expected for rats of this age and strain which are housed and treated under the conditions in this study. In addition, there was no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Female body weights and body weight gains of the 1000 mg/kg groups tended to be higher than the concurrent controls. This was statistically significant for group 6 females during gestation and lactation period. However, the gestation and lactation body weights and body weight gains remained within the historical control ranges and were therefore not considered treatment related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A slight, but statistically significant, increase in absolute and relative food consumption during gestation and lactation period was observed in Group 6, females only. This was not observed in Group 4 females, treated at the same dose level.
Overall, food consumption remained within normal limits and these findings were therefore not considered treatment related.
An isolated statistically significant difference, noted in females (higher relative food consumption at 200 mg/kg between post-coitum days 17-20), was regarded as chance finding, due to the lack of a dose-response relationship.

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In Group 4 females (1000 mg/kg), the mean number of platelets was statistically significantly lower compared to controls (relative difference 24%). This was mostly caused by one female, of which the number of platelets was below the P5-value of the historical control range, whereas the other females of the 1000 mg/kg group had normal numbers of platelets (close to the historical control mean). Therefore, the lower number of platelets of that one female was considered a chance finding and not to reflect an effect of the test item.
The statistically significantly lower number of total white blood cells noted in females at 200 mg/kg was regarded as unrelated to treatment due to the lack of a dose-related response.

Coagulation parameters (prothrombin time (PT) and activated partial thromboplastin time (APTT)) were unaffected by treatment. The mean APTT value of Group 4 females (1000 mg/kg) was reduced with 14% when compared to control (not statistically significant). This difference may be a change finding, related to the lower number of females for which this parameter was measured (n=3). In addition, the mean value remained well within the historical control range3. The decrease in APTT was therefore considered not to be treatment related. An isolated, statistically significant difference noted in males was regarded as unrelated to treatment due to the lack of a dose-related response (longer APTT at 200 mg/kg).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, the plasma concentrations of chloride (2% lower in males) and cholesterol (31 % higher in females) differed statistically significantly from the concurrent control values.
The mean chloride concentration in 1000 mg/kg males was at the lower end of the historical control range, mean cholesterol in 1000 mg/kg females remained within the normal range.

The statistically significant variations noted in clinical chemistry values at 40 and/or 200 mg/kg were regarded as unrelated to treatment due to the lack of a dose-related response (creatinine and sodium in males, alanine aminotransferase and inorganic phosphate in females).
Thyroid hormone analyses: Serum levels of T4 in males were comparable to concurrent control values.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Treated males had lower mean forelimb and hind limb grip strength values than controls (statistically significant except for hind limb grip strength at 200 mg/kg). The differences showed no dose-related response and grip strength of treated males remained in the normal range. Furthermore, the statistically significant differences could be attributed to the relatively high mean control value. Therefore, these findings were regarded as unrelated to treatment. Grip strength values in females were unremarkable.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
The motor activity in treated animals was comparable to controls. 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):
Liver weights (absolute and relative to body weight) were statistically significantly increased at 200 mg/kg in males and at 1000 mg/kg in both sexes. In males the microscopic correlate for this weight increase was hepatocellular hypertrophy.
Kidney weights (absolute and relative to body weights) were statistically significantly increased at 1000 mg/kg in males. The microscopic correlate for this kidney weight increase was hyaline droplet accumulation.

Any other differences, including those that reached statistical significance (i.e. heart weight and thyroid gland in males, thyroid gland and thymus weights in females) were considered not to be Ethyllinalyl Acetate-related due to the direction of the change, lack of dose-related pattern, and/or general overlap and variability in individual values.
Absolute mean kidney weight of Group 6 females (1000 mg/kg) was statistically significant increased when compared to concurrent control. As the relative kidney weight did not differ significantly from concurrent control, the increase in absolute kidney weight was attributed to the higher body weights of these females at necropsy.

Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Thymus:
Lymphoid atrophy was noted at a minimal degree in 3/5 females at 1000 mg/kg group.

Thyroid gland:
Mild follicular cell hypertrophy was noted in males at 1000 mg/kg. The minimal follicular cell hypertrophy observed in a few 40 and 200 mg/kg group males was considered to be a background finding. These incidences and severities were for example also observed in control males of a similar study type.

Liver:
Hepatocellular hypertrophy was noted at a minimal degree in 3/5 males at 1000 mg/kg.

Kidneys:
Hyaline droplet accumulation was noted at increased incidence and severity (5/5 moderate) in males at 1000 mg/kg. A background level of this finding (at minimal to slight degree) was present in 3/5 control males.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested. Item-test related microscopic findings at 1000mg/kg were considered as no adverse effects.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Analysis of dose preparations:

Accuracy: The concentrations analyzed in the formulations of Groups 2, 3, 4 and 6 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).

No test item was detected in the Group 1 and 5 formulations.

Homogeneity: The formulations of Groups 2, 4 and 6 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability: Formulations of Groups 2 and 4 were stable when stored at room temperature protected from light for at least four hours (i.e. relative difference over the storage period ≤10%).

Applicant's summary and conclusion

Conclusions:
In an oral OECD 422 screening study with rats, the NOAEL was determined to be at least 1000 mg/kg bw/day, based on no observed adverse effects at the highest dose tested.
Executive summary:

A combined 28d repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. Ethyllinalyl Acetate was administered by daily oral gavage to male and female rats at dose levels of 0, 40, 200 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 50-66 days).

Blood for toxicokinetic evaluation was sampled on treatment Day 10 (from pre-dose up to 24 hours post-dose). Blood was analysed for the concentrations of the test item and a defined metabolite i.e. Ethyllinalool. A toxicokinetic evaluation was performed.

No mortality was observed during the study. Few non-adverse test items-related findings at 200 mg/kg and 1000 mg/kg, such as salivation, lower chloride concentration (males) and higher cholesterol concentration (females). Increased liver weights in males and females were found. The increase of liver weight was considered an adaptive response, due to the induction of liver enzyme activities in both sexes. Increased kidney weight was associated to a hyaline droplet accumulation, most likely representing alpha 2 microglobuline. There was no indication of tubular damage, therefore these effects are considered no adverse. Moreover, this finding is specific for male rats and it is not relevant for humans. Mild effects were also observed in thymus and thyroids and are considered not adverse.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse

Effect levels (NOAELs) of Ethyllinalyl Acetate were established to be at least 1000 mg/ kg.