Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity - oral (OECD TG 422): NOAEL >= 500 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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.
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/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
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
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
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.
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.
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
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).
Key result
Critical effects observed:
no

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).

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is conducted according to methods similar to OECD guideline 422 and under GLP conditions. As such, it is considered appropriate to be used as the basis for the chemical safety assessment.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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.

Justification for classification or non-classification

Based on the available data, tetrahydrolinalyl acetate does not need to be classified for repeated dose toxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).