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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Dec 2017 - 21 Mar 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.
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:
05 Dec 2017 - 21 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
At current, 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.
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: See below at "Principles of method if other than guideline"
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following guidelines:
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016;
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000;
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008;
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008;
- EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 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 (incl. certificate)
Remarks:
22 JAN 2018
Limit test:
no
Species:
rat
Strain:
other:
Remarks:
Wistar Han
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 weeks (males) and 13 weeks (females)
- Weight at study initiation: 266 - 309 g (males), 202 - 238 g (females)
- Fasting period before study: no (F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.)
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). 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 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), ad libitum (except during motor activity measurements)
- Water: tap water, ad libitum (except during motor activity measurements)
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 38-59
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 05 Dec 2017 to 30 Jan 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
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.
- VEHICLE
- Justification for use and choice of vehicle (if other than water): Vehicle testing was performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 0, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in week 1 for concentration determination (all groups), homogeneity and stability (low and high dose groups). The homogeneity results obtained from the top, middle and bottom for the low and high dose group formulations were averaged and utilized as the concentration results. Stability of the test item in the vehicle upon storage for 4 hours at room
temperature protected from light. Analyses were performed using a validated analytical procedure.
Details on mating procedure:
After 15 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 the female that had not shown evidence of mating (this was the case for one control female) was separated from the male.
Duration of treatment / exposure:
Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13 or 15 days of lactation (for 50-56 days). Females without offspring or which had total litter loss were treated for 52 days (one non-mated control female) or 40-42 days.
Frequency of treatment:
Once daily
Duration of test:
Same as treatment.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 14-day dose range finder with oral gavage administration of Tetrahydrolinalyl Acetate in rats. The highest test dose, 500 mg/kg bw/day, was selected because testing of higher dose levels was considered unfeasible based on the substantial liver enlargement observed at 1000 mg/kg bw/day in the 14-day dose range finder (mean relative liver weights were increased by 41 and 56% in males and females, respectively).
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: on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal day 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal day 1, 4, 7, and 13.
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Subjective appraisal was maintained during the study
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (males only; females were not fasted)
- How many animals: all
- Parameters according to guidelines were examined (including thyroid hormone (T4) for F0-males only, prothrombin time and activated partial thromboplastin time)
- A blood smear was prepared from each haematology sample, analysis was only performed for one female in the low dose group.
URINALYSIS: No
FUNCTIONAL TESTS: Yes
- Time schedule for examinations: males during week 5 of treatment and the females during the last week of lactation (i.e. post natal days 6-13)
- Dose groups that were examined: all dose groups, 5 animals per group
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength (recorded as the mean of three measurements per animal) 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:
- Uterus weight: Yes
- Number of implantation sites: Yes
Fetal examinations:
- External examinations: Yes: all
STANDARDISATION OF LITTERS
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or anogenital distance, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable
PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number and sex of pups (on PND 1 and 4), stillbirths, live births, postnatal mortality (daily), presence of gross anomalies, weight gain (live pups were weighed individually on PND 1, 4, 7 and 13), physical or behavioural abnormalities (at least once daily), anogenital distance (for all live pups on PND 1, the AGD was normalized to the cube root of body weight.), presence of nipples/areolae in male pups (on post natal day 13).
GROSS EXAMINATION OF DEAD PUPS
In addition to one pup of a control litter that went missing during lactation, all pups of another control litter were found dead at first litter check. The five pups of this litter were examined externally, and their stomachs were examined for the presence of milk. The sex of these pups could not be determined due to cannibalism, therefore these pups were sexed as females. On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. 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. In addition, 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.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were con
ducted 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 le
ss than 3 observations. The following pairwise comparisons were made: Group 2 vs. Group 1, Group
3 vs. Group 1, Group 4 vs. Group 1.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared
using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a S
teel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-
Wallis. 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
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating - 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) x 100
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Nu
mber of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100
Historical control data:
Included in the report (where relevant)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs of toxicity were noted during detailed clinical observations or during weekly arena observations.
Salivation was noted at 500 mg/kg bw/day in all animals. 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), and was considered to be related to the irritancy of the test item. Any other clinical signs noted occurred incidentally and within the range of background findings to
be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be test item-related. One female rat dosed at 500 mg/kg bw/day died during anesthesia prior to blood sampling on the scheduled day of necropsy. This death was considered to be related to the blood sampling procedure and regarded as unrelated to the treatment with the test item. One control female was euthanized on Day 1 of lactation due to total litter loss.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and body weight gain were not affected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (before and after correction for body weight) was considered not to be affected by treatment. Higher mean food consumption values noted in females at 150 mg/kg bw/day during the postcoitum period (statistically significant between post-coitum Days 0-4 and 17-20) were regarded as unrelated to treatment due to the lack of a dose-related response.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment up to 500 mg/kg bw/day was not associated with changes in red blood cell parameters, white blood cell parameters or number of platelets. Isolated, statistically significant differences from control values were regarded as unrelated to treatment due to the lack of a dose-related response (mean corpuscular haemoglobin in females). Coagulation parameters (prothrombin time and activated partial thromboplastin time) were unaffected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical chemistry parameters. A statistically significant increase was noted in chloride levels of females at 500 mg/kg bw/day. As the difference between treated and control females was minimal (about 3%), the mean value remained well within historical control data and in the absence of a dose response, the increase was considered not toxicologically relevant. The statistically significant decrease noted in potassium levels of females at 50 mg/kg bw/day was considered unrelated to administration of the test item due to the lack of a dose-related response.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were noted for functional observations up to 500 mg/kg bw/day. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength were unaffected by treatment. The variation in motor activity did not indicaterelation with treatment. All treated groups showed a similar habituation profile with a decreasing trend
in activity over the duration of the test period. It was noted that all groups of treated males had higher mean values for total movements and ambulations than controls. The differences from controls were not statistically significant and showed no dose-related trend. Moreover, control values were lower than observed normally, particularly during intervals 1-3 and an abnormal habituation profile was obs
erved. As no differences were noted for the females and the results of the treated males were similar across different dose levels, the differences in motor activity in males were judged to be the result of slightly low concurrent control values rather than an effect of the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative liver weights were increased in females in a dose-related way (0%, 15% and 22% (absolute), 4%, 10% and 16% (relative) for the rats exposed to 50, 150 and 500 mg/kg bw/day, respectively). Kidney weights were affected in females, but in absence of dose-relationship this was considered not to be toxicologically significant (absolute weight: 2%, 10% and 9% increase compared to controls for groups exposed to 50, 150 and 500 mg/kg bw/day, respectively; relative weight: 6%, 6% and 3% increase compared to controls for groups exposed to 50, 150 and 500 mg/kg bw/day, respectively).
In addition, females at 500 mg/kg bw/day had lower weights of the thyroid gland, ovaries and uterus. The differences from control values (about 20%) were statistically significant, except for the absolute weights of the ovaries and uterus. All mean values remained within historical control data. There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There was one macroscopic finding which was possibly test item-related: an enlarged liver in one female of the 500 mg/kg bw/day group (microscopic correlate: cytoplasmic rarefaction, most likely glycogen storage).
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations in female rats.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to be affected by treatment. Except for one high dose female, which had an irregular cycle, all females had regular cycles, mostly of four days. The female with irregular cycle had a normal litter. The occurrence of one irregular cycle in a single treated female, without an apparent correlation to pregnancy status, was considered not to reflect an effect of the test item.
Number of abortions:
no effects observed
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 85, 90, 85 and 94% for the control, 50, 150 and 500 mg/kg groups, respectively. The post-implantation survival indices for the control and 150 mg/kg groups were slightly lower than normal. This could be explained by (slightly) high numbers of unaccounted for sites in a few females (7/12 for one control female; 5/13 and 5/14 for two females dosed at 150 mg/kg bw/day). The slightly high numbers of unaccounted for sites in two females at 150 mg/kg bw/day were regarded as unrelated to treatment due to the lack of a dose-related trend.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The number of living pups at first litter check (live litter size) was not affected by treatment. Live birth index was not affected by treatment. The live birth indices were 100% for all test item groups and 94% for the control group. At first litter check, all five pups of one control female were found dead (total litter loss). The occurrence of total litter loss in a single female was within normal limits.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation index and duration of gestation were considered not to be affected by treatment. Except for one control female (only dead pups at first litter check) and one female at 500 mg/kg bw/day (only one implantation site), all pregnant females had live offspring. The gestation indices were 88, 100, 100 and 90% for the control, 50, 150 and 500 mg/kg bw/day groups, respectively. These cases of failed pregnancy occurred without related histopathology changes in reproductive organs. The incidental failed pregnancy at 500 mg/kg was regarded as unrelated to treatment.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
Fertility index was considered not to be affected by treatment. Except for one control female and two females at 150 mg/kg bw/day, all females with evidence of mating were pregnant. The fertility indices were 89, 100, 80 and 100% for the control, 50, 150 and 500 mg/kg bw/day groups, respectively.
These cases of non-pregnancy occurred without related histopathology changes in reproductive organs. The non-pregnancies at 150 mg/kg were regarded as unrelated to treatment due to the lack of a dose-related trend.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects seen up to and including the highest dose tested (500 mg/kg bw/day)
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
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 100% for all test item groups and 99% for the control group.
One pup of the control group went missing (presumed cannibalized) at PND 2. This incidental pup mortality was within normal limits.
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment. Sex ratio at 150 mg/kg bw/day differed statistically significantly from the control ratio (percentage of males/females: 63/37 versus 45/55). To a lesser extent, sex ratio was also skewed at 50 mg/kg (males/females: 60/40). The sex ratios in these test item groups were at the upper end of the historical control range. In the absence of a dose-related response (sex ratio at 500 mg/kg was 58/42) these intergroup differences were judged to be unrelated to treatment with the test item.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
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. No pups died after PND 4, resulting in a lactation index of 100% for all groups.
External malformations:
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment. Treatment up to 500 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13. No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of most findings noted remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment. An unusual finding, accessory tissue at the corner(s) of the mouth, was noted in a pup of the 150 mg/kg bw/day group. As only a single treated pup was affected and a similar abnormality occurred in a control pup, this finding was regarded as unrelated to treatment.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects seen up to and including the highest dose tested (500 mg/kg bw/day)
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Conclusions:
In an oral repeated dose study with screening for reproduction and developmental toxicity (OECD 422 guideline study) with rats, the parental NOAEL of Tetrahydrolinalyl Acetate was determined to be 500 mg/kg bw/day, based on the fact that no adverse effects were seen at the highest dose tested.
Executive summary:

A combined 28d repeated dose study with screening for reproductive and developmental effects was performed according to OECD/EC guidelines and GLP principles. Tetrahydrolinalyl Acetate was administered by daily oral gavage to male and female rats at dose levels of 50, 150 and 500 mg/kg bw/ day. Females that delivered offspring were exposed for 2 weeks prior to mating, during mating, during post-coitum, and 13 or 15 days of lactation (for 50-56 days). Females without offspring or which had total litter loss were treated for 52 days (one non-mated control female) or 40-42 days. Formulation analysis demonstrated acceptable accuracy and homogeneity of the formulations. Treatment up to 500 mg/kg bw/day was well tolerated as indicated by the absence of adverse changes in the parental parameters examined (i.e. survival, clinical appearance, functional observations, body weight, food consumption, hematology, clinical chemistry (including serum T4 in males), macroscopic examination, organ weights, and microscopic examination). No reproduction or developmental toxicity was observed up to the highest dose level tested (500 mg/kg bw/day).

Based on the fact that no adverse effects were seen at the highest dose tested, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of 500 mg/kg bw/day was established.

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:
05 Dec 2017 - 21 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
At current, 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:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: See below at "Principles of method if other than guideline"
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following guidelines:
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016;
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000;
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008;
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008;
- EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 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 (incl. certificate)
Remarks:
22 JAN 2018
Limit test:
no
Species:
rat
Strain:
other:
Remarks:
Wistar 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. The total number of animals
used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 weeks (males and 13 weeks (females)
- Weight at study initiation: 266 - 309 g (males), 202 - 238 g (females)
- Fasting period before study: no (F0-males were fasted overnight with a maximum of 24 hours be fore blood sampling, but water was available. F0-females were not fasted overnight.)
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). 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 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), ad libitum (except during motor activity measurements)
- Water: tap water, ad libitum (except during motor activity measurements)
- Acclimation period: 6 days
DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 38-59
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From 05 Dec 2017 to 30 Jan 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
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.
- VEHICLE
- Justification for use and choice of vehicle (if other than water): Vehicle testing was performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 0, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Details on mating procedure:
After 15 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 the female that had not shown evidence of mating (this was the case for one control female) was separated from the male.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in week 1 for concentration determination (all groups), homogeneity and stability (low and high dose groups). The homogeneity results obtained from the top, middle and bottom for the low and high dose group formulations were averaged and utilized as the concentration results. Stability of the test item in the vehicle upon storage for 4 hours at room temperature protected from light.
Analyses were performed using a validated analytical procedure.
Duration of treatment / exposure:
Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days) . Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13 or 15 days of lactation (for 50-56 days). Females without offspring or which had total litter loss were treated for 52 days (one non-mated control female) or 40-42 days.
Frequency of treatment:
Once daily.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 14-day dose range finder with oral gavage administration of Tetrahydrolinalyl Acetate in rats. The highest test dose, 500 mg/kg bw/day, was selected because testing of higher dose levels was considered unfeasible based on the substantial liver enlargement observed at 1000 mg/kg bw/day in the 14-day dose range finder (mean relative liver weights were increased by 41 and 56% in males and females, respectively).
Positive control:
Not included
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: on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal day 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post
natal day 1, 4, 7, and 13.
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Subjective appraisal was maintained during the study
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (males only; females were not fasted)
- How many animals: all
- Parameters according to guidelines were examined (including thyroid hormone (T4) for F0-males only, prothrombin time and activated partial thromboplastin time)
- A blood smear was prepared from each haematology sample, analysis was only performed for one female in the low dose group.
URINALYSIS: No
FUNCTIONAL TESTS: Yes
- Time schedule for examinations: males during week 5 of treatment and the females during the last week of lactation (i.e. post natal days 6-13)
- Dose groups that were examined: all dose groups, 5 animals per group
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength (recorded as the mean of three measurements per animal) 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 (pre test 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 terminat
ion of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.
Sperm parameters (parental animals):
The testes of males that failed to sire and testes of males which were selected were histopathologically examined, including spermatogenic staging.
Litter observations:
STANDARDISATION OF LITTERS
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or anogenital distance, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable

PARAMETERS EXAMINED
The following parameters were examined in offspring:
Number and sex of pups (on PND 1 and 4), stillbirths, live births, postnatal mortality (daily), presence of gross anomalies, weight gain (live pups were weighed individually on PND 1, 4, 7 and 13), physical or behavioural abnormalities (at least once daily), anogenital distance (for all live pups on PND 1, the AGD was normalized to the cube root of body weight.), presence of nipples/areolae in male pups (on post natal day 13).

Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The main organs were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body we
ight) were calculated.
HISTOPATHOLOGY: Yes
Histology was performed on main organs and tissues.
In addition:
Males that failed to sire (except for males which were selected) and females that failed to deliver pups: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina. Female with total litter loss: Mammary gland.
Remaining animals: Gross lesions/masses.
Postmortem examinations (offspring):
METHOD OF EUTHANASIA
Pups, younger than 7 days were euthanized by decapitation. All remaining 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 EXAMINATION OF DEAD PUPS
In addition to one pup of a control litter that went missing during lactation, all pups of another control litter were found dead at first litter check. The five pups of this litter were examined externally, and their stomachs were examined for the presence of milk. The sex of these pups could not be determined due to cannibalism, therefore these pups were sexed as females.

On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. 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. In addition, 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.
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.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. 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.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- 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) x 100
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100
- 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):
No test item-related clinical signs of toxicity were noted during detailed clinical observations or during weekly arena observations.
Salivation was noted at 500 mg/kg bw/day in all animals. 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), and was considered to be related to the irritancy of the test item. Any other clinical signs noted occurred incidentally and within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be test item-related. One female rat dosed at 500 mg/kg bw/day died during anesthesia prior to blood sampling on the scheduled day of necropsy. This death was considered to be related to the blood sampling procedure and
regarded as unrelated to the treatment with the test item. One control female was euthanized on Day 1 of lactation due to total litter loss.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and body weight gain were not affected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (before and after correction for body weight) was considered not to be affected by treatment. Higher mean food consumption values noted in females at 150 mg/kg bw/day during the postcoitum period (statistically significant between post-coitum Days 0-4 and 17-20) were regarded
as unrelated to treatment due to the lack of a dose-related response.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment up to 500 mg/kg bw/day was not associated with changes in red blood cell parameters, white blood cell parameters or number of platelets. Isolated, statistically significant differences from control values were regarded as unrelated to treatment due to the lack of a dose-related response (hemoglobin and hematocrit in males, mean corpuscular haemoglobin in females). Coagulation parameters (prothrombin time and activated partial thromboplastin time) were unaffected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical chemistry parameters. A statistically significant decrease was noted in sodium levels of males at 150 and 500 mg/kg bw/day. Compared to concurrent controls, mean sodium levels were about 17% decreased at both dose levels. However, as the mean values remained well within historical control data, the decrease was considered not toxicologically relevant. A statistically significant increase was noted in chloride levels of females at 500 mg/kg bw/day. As the difference between treated and control females was minimal (about 3%), the mean value remained well within historical control data and in the absence of a dose response, the increase was considered not toxicologically relevant. The statistically significant decrease noted in potassium levels of females at 50 mg/kg bw/day was considered unrelated to administration of the test item due to the lack of a dose-related response. Serum levels of T4 in F0 males were not affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were noted for functional observations up to 500 mg/kg bw/day. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength were unaffected by treatment. The variation in motor activity did not indicate a relation with treatment. All treated groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. It was noted that all groups of treated males had higher mean values for total movements and ambulations than controls. The differences from controls were not statistically significant and showed no dose-related trend. Moreover, control values were lower than observed normally, particularly during intervals 1-3 and an abnormal habituation profile was observed. As no differences were noted for the females and the results of the treated males were similar across different dose levels, the differences in motor activity in males were judged to be the result of slightly low concurrent control values rather than an effect of the test item.
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):
There were no test item-related microscopic observations in female rats. In male rats, test item-related microscopic findings were noted in the liver at 500 mg/kg bw/day (centrilobular hepatocellular hypertrophy at a minimal degree in 3/5 males, not observed in any of the
other groups). Based on the modest magnitude of the increase in liver weight (about 20%) and the absence of any degenerative, inflammatory or proliferative findings, the observed hepatic effects were regarded as non-adverse. In the kidneys starting at 50 mg/kg bw/day (hyaline droplets accumulation in controls: 2/5 minimal; low dose: 4/5 minimal, 1/5 slight; mid dose: 1/5 minimal, 3/5 slight; high dose: 5/5 moderate). The hyaline droplet accumulation likely represented alpha2u-globulin, a normal protein in male rats which un
dergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury. This male rat specific protein is not present in female rats nor in higher mammals, including man. The increased hyaline droplet accumulation observed in males exposed to the test item was not accompanied by indicators of
renal tubular damage and was therefore considered to be a non-adverse microscopic finding. There were no other test item-related histologic changes. There was one finding of note: At macroscopic examination, a greenish, hard nodule (18x8 mm) was noted in the mammary gland (left axillary region) of one high dose female. At microscopy this appeared to be an adenocarcinoma, a neoplastic
lesion that can be seen as a background finding in rats of this age and strain. The neoplastic lesion in this female and 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:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to be affected by treatment. Except for one high dose female, which had an irregular cycle, all females had regular cycles, mostly of four days. The female with irregular cycle had a normal litter. The occurrence of one irregular cycle in a single treated female, without an apparent correlation to pregnancy status, was considered not to reflect an effect of the test item.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for test item-related abnormal spermatogenesis.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Gestation index and duration of gestation were considered not to be affected by treatment. Except for one control female (only dead pups at first litter check) and one female at 500 mg/kg bw/day (only one implantation site), all pregnant females had live offspring. The gestation indices were 88, 100, 100 and 90% for the control, 50, 150 and 500 mg/kg bw/day groups, respectively. These cases of failed pregnancy occurred without related histopathology changes in reproductive organs. The incidental failed pregnancy at 500 mg/kg was regarded as unrelated to 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. 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 85, 90, 85 and 94% for the control, 50, 150 and 500 mg/kg groups, respectively.
The post-implantation survival indices for the control and 150 mg/kg groups were slightly lower than normal. This could be explained by (slightly) high numbers of unaccounted for sites in a few females (7/12 for one control female; 5/13 and 5/14 for two females dosed at 150 mg/kg bw/day). The slightly high numbers of unaccounted for sites in two females at 150 mg/kg bw/day were regarded as unrelated to treatment due to the lack of a dose-related trend.
The number of living pups at first litter check (live litter size) was not affected by treatment.
Live birth index was not affected by treatment. The live birth indices were 100% for all test item groups and 94% for the control group. At first litter check, all five pups of one control female were found dead (total litter loss). The occurrence of total litter loss in a single female was within normal limits.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects seen at highest dose tested (500 mg/kg bw/day)
Key result
Critical effects observed:
no
Remarks on result:
not measured/tested
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. Most of the clinical signs observed remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment. A few unusual findings noted in a pup of the 150 mg/kg group, consisting of a swollen under lip (at PND 13-15), cloudy/turbid eyes (at PND 15-16) and accessory tissue at the corners of the mouth (at last litter check on PND16). These abnormalities did not occur in any other pups of females administered the test item. Moreover, accessory tissue at a corner of the mouth was also noted in a control pup. Therefore, the unusual findings in a single 150 mg/kg bw/day pup were considered not to be test item-related.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
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 100% for all test item groups and 99% for the control group. One pup of the control group went missing (presumed cannibalized) at PND 2. This incidental pup mortality was within normal limits.
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. No pups died after PND 4, resulting in a lactation index of 100% for all groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects seen at the highest dose tested (500 mg/kg bw/day)
Key result
Critical effects observed:
no
Remarks on result:
not measured/tested
Key result
Reproductive effects observed:
no

The results of the dose range finding study, the formulation analyses and the toxicokinetic assessment are included in section 7.5.1.

Conclusions:
In an oral repeated dose study with screening for reproduction and developmental toxicity (OECD 422 guideline study) with rats, the parental, reproduction and developmental NOAEL of Tetrahydrolinalyl Acetate was determined to be 500 mg/kg bw/day, based on no adverse effects seen at the highest dose tested.
Executive summary:

A combined 28d repeated dose study with screening for reproductive and developmental effects was performed according to OECD/EC guidelines and GLP principles. Tetrahydrolinalyl Acetate was administered by daily oral gavage to male and female rats at dose levels of 50, 150 and 500 mg/kg bw/ day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were exposed for 2 weeks prior to mating, during mating, during post-coitum, and 13 or 15 days of lactation (for 50-56 days). Females without offspring or which had total litter loss were treated for 52 days (one non-mated control female) or 40-42 days. Formulation analysis demonstrated acceptable accuracy and homogeneity of the formulations. Treatment up to 500 mg/kg bw/day was well tolerated as indicated by the absence of adverse changes in the parental parameters examined (i.e. survival, clinical appearance, functional observations, body weight, food consumption, hematology, clinical chemistry (including serum T4 in males), macroscopic examination, organ weights, and microscopic examination). No reproduction or developmental toxicity was observed up to the highest dose level tested (500 mg/kg bw/day).

Based on the fact that no adverse effects were seen at the highest dose tested, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of 500 mg/kg bw/day was established.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: See below at "Principles of method if other than guideline"
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following guidelines:
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016;
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000;
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008;
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008;
- EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 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 (incl. certificate)
Remarks:
22 JAN 2018
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Clear liquid
Details on test material:
Test item storage: At room temperature protected from light, container flushed with nitrogen, desiccated
Test item handling: Use amber glassware or wrap container in aluminum-foil

Test animals

Species:
rat
Strain:
other:
Remarks:
Wistar 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. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 weeks (males and 13 weeks (females)
- Weight at study initiation: 266 - 309 g (males), 202 - 238 g (females)
- Fasting period before study: no (F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.)
- Housing: On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, males were housed in their home cage with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages. During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). 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 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), ad libitum (except during motor activity measurements)
- Water: tap water, ad libitum (except during motor activity measurements)
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water is performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22
- Humidity (%): 38-59
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 05 Dec 2017 to 30 Jan 2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
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.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Vehicle testing was performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 0, 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in week 1 for concentration determination (all groups), homogeneity and stability (low and high dose groups). The homogeneity results obtained from the top, middle and bottom for the low and high dose group formulations were averaged and utilized as the concentration results. Stability of the test item in the vehicle upon storage for 4 hours at room temperature protected from light.
Analyses were performed using a validated analytical procedure.
Duration of treatment / exposure:
Males were treated for two weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for two weeks prior to mating, during mating, during post-coitum, and 13 or 15 days of lactation (for 50-56 days). Females without offspring or which had total litter loss were treated for 52 days (one non-mated control female) or 40-42 days.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 14-day dose range finder with oral gavage administration of Tetrahydrolinalyl Acetate in rats. The highest test dose, 500 mg/kg bw/day, was selected because testing of higher dose levels was considered unfeasible based on the substantial liver enlargement observed at 1000 mg/kg bw/day in the 14-day dose range finder (mean relative liver weights were increased by 41 and 56% in males and females, respectively).
Positive control:
Not included.

Examinations

Observations and examinations performed and frequency:
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: on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal day 1, 4, 7, and 13. Terminal body weights were recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on post natal day 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Subjective appraisal was maintained during the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (males only; females were not fasted)
- How many animals: all
- Parameters according to guidelines were examined (including thyroid hormone (T4) for F0-males only, prothrombin time and activated partial thromboplastin time)
- A blood smear was prepared from each haematology sample, analysis was only performed for one female in the low dose group.

URINALYSIS: No

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: males during week 5 of treatment and the females during the last week of lactation (i.e. post natal days 6-13)
- Dose groups that were examined: all dose groups, 5 animals per group
- Battery of functions tested: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (total movements and ambulations)

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The main organs were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes
Histology was performed on main organs and tissues.
In addition:
Males that failed to sire (except for males which were selected) and females that failed to deliver pups: Cervix, epididymis, coagulation gland, prostate gland,
seminal vesicles, ovaries, testes, uterus and vagina. Female with total litter loss: Mammary gland. Remaining animals: Gross lesions/masses.
Other examinations:
BIOANALYSIS AND TOXICOKINETIC EVALUATION
On Day 10 of treatment, blood samples (~0.4 mL using K2-EDTA tubes) were collected from the jugular vein at various timepoints after dosing. Animals were restrained during blood collection.
Samples were analyzed for the concentration of test item and the specified metabolite Tetrahydrolinalool using a validated GC-MS method.
Toxicokinetic parameters were estimated using Phoenix pharmacokinetic software. A noncompartmental approach consistent with the oral route of administration was used for parameter estimation. All parameters were generated from Tetrahydrolinalyl Acetate (THLAc) and Tetrahydrolinalool (THL) using composite concentrations in plasma. Parameters that were calculated included tlast, tmax, Cmax, AUClast, dose effect, sex effect and exposure to metabolite versus parent. No log-linear regression was possible, as a consequence the half-life could not be calculated.
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.
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.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, treatment-related
Description (incidence and severity):
No test item-related clinical signs of toxicity were noted during detailed clinical observations or during weekly arena observations.
Salivation was noted at 500 mg/kg bw/day in all animals. 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), and was considered to be related to the irritancy of the test item.
Any other clinical signs noted occurred incidentally and within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period that was considered to be test item-related. One female rat dosed at 500 mg/kg bw/day died during anesthesia prior to blood sampling on the scheduled day of necropsy. This death was considered to be related to the blood sampling procedure and regarded as unrelated to the treatment with the test item. One control female was euthanized on Day 1 of lactation due to total litter loss.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and body weight gain were not affected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption (before and after correction for body weight) was considered not to be affected by treatment. Higher mean food consumption values noted in females at 150 mg/kg bw/day during the postcoitum period (statistically significant between post-coitum Days 0-4 and 17-20) were regarded as unrelated to treatment due to the lack of a dose-related response.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment up to 500 mg/kg bw/day was not associated with changes in red blood cell parameters, white blood cell parameters or number of platelets. Isolated, statistically significant differences from control values were regarded as unrelated to treatment due to the lack of a dose-related response (hemoglobin and hematocrit in males, mean corpuscular haemoglobin in females). Coagulation parameters (prothrombin time and activated partial thromboplastin time) were unaffected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical chemistry parameters. A statistically significant decrease was noted in sodium levels of males at 150 and 500 mg/kg bw/day. Compared to concurrent controls, mean sodium levels were about 17% decreased at both dose levels. However, as the mean values remained well within historical control data, the decrease was considered not toxicologically relevant.
A statistically significant increase was noted in chloride levels of females at 500 mg/kg bw/day. As the difference between treated and control females was minimal (about 3%), the mean value remained well within historical control data and in the absence of a dose response, the increase was considered not toxicologically relevant. The statistically significant decrease noted in potassium levels of females at 50 mg/kg bw/day was considered unrelated to administration of the test item due to the lack of a dose-related response. Serum levels of T4 in F0 males were not affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were noted for functional observations up to 500 mg/kg bw/day. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Forelimb and hind limb grip strength were unaffected by treatment. The variation in motor activity did not indicate a relation with treatment. All treated groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. It was noted that all groups of treated males had higher mean values for total movements and ambulations than controls. The differences from controls were not statistically significant and showed no dose-related trend. Moreover, control values were lower than observed normally, particularly during intervals 1-3 and an abnormal habituation profile was observed. As no differences were noted for the females and the results of the treated males were similar across different dose levels, the differences in motor activity in males were judged to be the result of slightly low concurrent control values rather than an effect of the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative liver weights were increased in males and females in a dose-related way (males: 4%, 10% and 19% (absolute), 4%, 9% and 18% (relative); females: 0%, 15% and 22% (absolute), 4%, 10% and 16% (relative) for the rats exposed to 50, 150 and 500 mg/kg bw/day, respectively.
There were test item-related increases in the weights of the kidneys in males (absolute weight: 10% and 13% increase compared to controls for groups exposed to 150 and 500 mg/kg bw/day, respectively; relative weight: 2%, 8% and 13% increase compared to controls for groups exposed to 50, 150 and 500 mg/kg bw/day, respectively). Kidney weights were also affected in females, but in absence of dose-relationship this was considered not to be toxicologically significant (absolute weight: 2%, 10% and 9% increase compared to controls for groups exposed to 50, 150 and 500 mg/kg bw/day, respectively; relative weight: 6%, 6% and 3% increase compared to controls for groups exposed to 50, 150 and 500 mg/kg bw/day, respectively).
In addition, females at 500 mg/kg bw/day had lower weights of the thyroid gland, ovaries and uterus. The differences from control values (about 20%) were statistically significant, except for the absolute weights of the ovaries and uterus. All mean values remained within historical control data. There were no other test item-related organ weight changes.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There was one macroscopic finding which was possibly test item-related: an enlarged liver in one female of the 500 mg/kg bw/day group (microscopic correlate: cytoplasmic rarefaction, most likely glycogen storage).
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations in female rats.
In male rats, test item-related microscopic findings were noted in the liver at 500 mg/kg bw/day (centrilobular hepatocellular hypertrophy at a minimal degree in 3/5 males, not observed in any of the other groups). Based on the modest magnitude of the increase in liver weight (about 20%) and the absence of any degenerative, inflammatory or proliferative findings, the observed hepatic effects were regarded as non-adverse.
In the kidneys starting at 50 mg/kg bw/day (hyaline droplets accumulation in controls: 2/5 minimal; low dose: 4/5 minimal, 1/5 slight; mid dose: 1/5 minimal, 3/5 slight; high dose: 5/5 moderate). The hyaline droplet accumulation likely represented alpha2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury. This male rat specific protein is not present in female rats nor in higher mammals, including man. The increased hyaline droplet accumulation observed in males exposed to the test item was not accompanied by indicators of renal tubular damage and was therefore considered to be a non-adverse microscopic finding.
There were no other test item-related histologic changes. There was one finding of note: At macroscopic examination, a greenish, hard nodule (18x8 mm) was noted in the mammary gland (left axillary region) of one high dose female. At microscopy this appeared to be an adenocarcinoma, a neoplastic lesion that can be seen as a background finding in rats of this age and strain.
The neoplastic lesion in this female and 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:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
500 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 (500 mg/kg bw/day).

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Results of the 14-day dose range finding study:

In the range finder dose levels of 250, 500 and 1000 mg/kg bw/day were tested. Treatment-related slight to moderate salivation was seen immediately after dosing until 1 hour after dosing in all treated animals, especially in animals dosed with 500 or 1000 mg/kg bw/day. A treatment-related change of the liver of males was observed at macroscopic examinations; 1/5, 3/5 and 5/5 males treated at 250, 500 and 1000 mg/kg bw/day, respectively, showed an enlarged liver. This correlated with a treatment-related statistically significant increase in relative liver weights of males. The mean relative liver weight of males was 13%, 29% and 41% higher compared to controls at 250, 500 and 1000 mg/kg bw/day, respectively. For females higher relative liver weights were noted as well. The mean relative liver weights

were 21%, 34% and 56% higher compared to controls at 250, 500 and 1000 mg/kg bw/dy, respectively. In females, no correlating macroscopic findings were noted in the liver. Therefore, based on the magnitude of the increase in relative liver weights (56% higher than

concurrent controls) in females after short-term treatment (two weeks only) with 1000 mg/kg, this dose level was considered unfeasible for the main study.

Dose Formulation Analyses:

Accuracy: The concentrations analyzed in the formulations of the low and the high dose groups were in agreement with target concentrations (i.e. mean accuracies between 89% and 95%). The mean concentration measured in the mid dose group formulation was 89% of the target value. This minor deviation from the target concentration was considered acceptable and without impact on the study results. No test item was detected in the control group formulation.

Homogeneity: The formulations of the mid and high dose groups were homogeneous (i.e. coefficient of variation 4.7% and 6.3%).

Stability: Formulations of the mid and the high dose groups were stable when stored at room temperature protected from light for at least 4 hours (i.e. relative difference over the storage period: -0.52 - 5.0%).

Results toxicokinetic assessment:

Toxicokinetic analyses, conducted on male and female rats after 10 days of treatment, showed that all blood concentrations of the test item and its metabolite Tetrahydrolinalool in the control animals were below the detection limit (5.00 ng/mL).

In treated animals, the following observations were made:

- The blood of all treated rats contained test item, indicating that the test item was absorbed from the gastrointestinal tract. The metabolite could be detected only in rats of the 500 mg/kg bw/day group (between 1-8 h post-dose in females and at 4 h post-dose in males).

- Blood concentrations of the test item increased slowly. The peak blood concentration, Cmax, was reached at 2-4 h. Tlast ranged from 4 to 24 h post-dose and increased with increasing dose levels. Cmax of the metabolite was reached at 4 h post-dose at 500 mg/kg bw/day in females.

- Peak concentrations of the test item increased dose-proportionally over the dose range tested (50-500 mg/kg bw/day), except in females from 50 to 150 mg/kg bw/day and from 50 to 500 mg/kg bw/day where the increase was more than dose-proportional.

- The systemic exposure, expressed as AUClast of the test item, increased with increasing dose in a slightly more than dose-proportional manner over the dose range tested, except in males from 150 to 500 mg/kg bw/day where the increase was dose-proportional.

- Systemic exposure to the test item showed no clear sex differences at 50 and 150 mg/kg bw/day. At 500 mg/kg bw/day, systemic exposure showed a sex difference for AUClast (about two-fold higher in females than in males) but not for Cmax.

- Systemic exposure to the metabolite, in terms of Cmax and AUClast, was about 100 times lower than that to the unchanged test item (this comparison could only be made in females of the 500 mg/kg bw/day group as in males treated at 500 mg/kg bw/day and in males and females treated at 50 or 150 mg/kg bw/day the blood concentrations of Tetrahydrolinalool were generally below the lower limit of quantification).

Applicant's summary and conclusion

Conclusions:
In an oral repeated dose study with screening for reproduction and developmental toxicity (OECD 422 guideline study) with rats, the parental NOAEL of Tetrahydrolinalyl Acetate was determined to be 500 mg/kg bw/day, based on the fact that no adverse effects were seen at the highest dose tested.
Executive summary:

A combined 28d repeated dose study with screening for reproductive and developmental effects was performed according to OECD/EC guidelines and GLP principles. Tetrahydrolinalyl Acetate was administered by daily oral gavage to male and female rats at dose levels of 50, 150 and 500 mg/kg bw/ day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were exposed for 2 weeks prior to mating, during mating, during post-coitum, and 13 or 15 days of lactation (for 50-56 days). Females without offspring or which had total litter loss were treated for 52 days (one non-mated control female) or 40-42 days. Formulation analysis demonstrated acceptable accuracy and homogeneity of the formulations. Treatment up to 500 mg/kg bw/day was well tolerated as indicated by the absence of adverse changes in the parental parameters examined (i.e. survival, clinical appearance, functional observations, body weight, food consumption, hematology, clinical chemistry (including serum T4 in males), macroscopic examination, organ weights, and microscopic examination). Slight salivation after dosing occurred at 500 mg/kg bw/day in all animals. This was regarded as a physiological response related to the irritant properties of the test item rather than a sign of systemic toxicity. Liver weight (absolute and relative to body weight) was increased at 500 mg/kg bw/day in both sexes. Microscopically, this correlated with minimal centrilobular hepatocellular hypertrophy in males (there was no microscopic correlate in females). Based on the modest magnitude of the increase in liver weight (about 20%) and the absence of any degenerative, inflammatory or proliferative findings, the observed hepatic effects were regarded as non-adverse. Microscopic examination showed a test item-related increase in the incidence and severity (up to moderate degree) of hyaline droplet accumulation in the kidneys of male rats starting at 50 mg/kg bw/day (with correlating increased renal weight at 500 mg/kg bw/day). The hyaline droplet accumulation likely represented alpha2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury. This male rat specific protein is not present in female rats nor in higher mammals, including man. The increased hyaline droplet accumulation observed in males exposed to the test item was not accompanied by indicators of renal tubular damage and was therefore considered to be a non-adverse microscopic finding. Lower weights of the thyroid, ovaries and uterus were noted in females at 500 mg/kg bw/day (about 20% difference from control values). These organ weight changes remained within historical control data and occurred in the absence of histopathological alterations and were, therefore, regarded as non-adverse.

Based on the fact that no adverse effects were seen at the highest dose tested, a parental No Observed Adverse Effect Level (NOAEL) of 500 mg/kg bw/day was established.