Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity - oral (OECD 422): NOAEL >= 1000 mg/kg bw/day

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 dec 2017 - 03 dec 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Justification for type of information:
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)
Version / remarks:
July 2000
Deviations:
no
Principles of method if other than guideline:
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016);
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)
- Guideline on Bioanalytical Method Validation, European Medicines Agency (EMA), EMEA/CHMP / EWP/192217/2009, 21 July 2011
- Bioanalytical Method Validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER) and Center for Veterinary Medicine (CVM), May 2001
- ICH M3 (R2). Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals. December 2009
- ICH S3a. Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies, October 1994.
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
No correction factor required
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males) and 13 weeks (females)
- Weight at study initiation: 261 - 295 g (males) and 209 and 241 g (females)
- Fasting period before study: No
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in Macrolon cages.
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon cages.
During the post-mating phase, males were housed in Macrolon cages with a maximum of 5 males/cage. Females were individually housed in Macrolon cages.
During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups
were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), adlibitum . The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water: tap water, ad libitum. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water was performed.
- Acclimation period: 5 days

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

IN-LIFE DATES: From 12 Dec 2017 to 27 Jul 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily as a solution and dosed within 4 hours after adding the test item to the vehicle protected from light.
The test item was directly mixed with the vehicle to prevent any loss; therefore vehicle was weighed prior to the test item.
Test item dosing formulations were kept at room temperature protected from light until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Dose volume: 5 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure.
Stability: During the course of this study at one occasion during the treatment phase of Group 1-4, stability of the prepared formulation was determined at 4 hours at room temperature under protection from light. Duplicate sets of each sample (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation.
Homogeneity: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.
Concentration: Duplicate sets of samples (approximately 500 mg accurately weighed) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions for suspensions of target concentration.
Duration of treatment / exposure:
- Males were exposed for 29-30 days, i.e. 2 weeks prior to mating, during mating, and up to termination.
- Females were exposed for 50-56days, 62 days (one of Group 1 and one of Group 2) or 66 days (one of Group 5) i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day before scheduled necropsy.
- Females which failed to deliver (non-pregnant or implantation site only) were treated for 41 or 43 days, and one non-mated female was treated for 52 days.
- Four females were not dosed on one occasion as these females were littering at the time of dosing.
Frequency of treatment:
Once daily, 7 d/w
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
This group was added to the study because the evaluation of pup body weight at PND 13 and nipple retention were determined for an insufficient number of litters in Group 1-4 or three litters (1000 mg/kg)). This was considered insufficient to provide conclusive evidence of absence of an effect of treatment and therefore Groups 5 (Control) and 6 (1000 mg/kg) were included to complete this evaluation
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected based on the results of a 14-day dose range finder with oral gavage administration of Ethyllinalyl Acetate in rats.
In the range finder dose levels of 250, 500 and 1000 mg/kg were tested.
- Treatment-related slight to moderate salivation was seen in animals treated at 1000 mg/kg.
- At 500 and 250 mg/kg slight salivation was observed. A treatment-related change of the liver of males was observed at macroscopic examinations; 1/5, 4/5 and 5/5 males treated at 250, 500 and 1000 mg/kg, respectively, showed an enlarged liver. This correlated with a treatment-related statistically significant increase in relative liver weights of males. After 14 days of exposure, the enzymatic activities were significantly increased for the PROD, BROD and HCG activities at a dose level of 1000 mg/kg. No significant increase was noted for the EROD activities in any of the tested dose levels.
- For females, higher relative liver weights were noted as well. The mean relative liver weights were 13% and 28% higher compared to controls at 500 and 1000 mg/kg, respectively (statistically significant at 1000 mg/kg only). The enzymatic activities were significantly increased for the EROD, PROD and HCG activities at the dose levels of 500 mg/kg and 1000 mg/kg and for the BROD activities at the dose level of 1000 mg/kg.
The increased liver weights at 1000 mg/kg were considered acceptable to set the high dose at 1000 mg/kg for the main study. Based on the increase in relative liver weight in males at 500 mg/kg a spacing factor of five was used for dose level selection.

As dose levels for the main study 40, 200 and 1000 mg/kg were selected.
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: Twice daily.

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

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

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

FOOD EFFICIENCY:
No.

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

OPHTHALMOSCOPIC EXAMINATION: No

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

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

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

URINALYSIS: No

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

IMMUNOLOGY: No


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

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

Statistics:
- All statistical tests were conducted at the 5% significance level.
- All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
- Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion.
- Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
- Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
- The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1,
Group 4 vs. Group 1, and Group 6 vs. Group 5.
- Parametric: Datasets with at least 2 groups (the designated control group and 1 other group) were compared using Dunnett-test (many-to-one-t-test).
- Non-Parametric: Datasets with at least 2 groups was compared using a Steel-test (many-to-one rank test).
- The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence: an overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was noted at 200 and 1000 mg/kg with increasing frequency. At 1000 mg/kg all animals showed salivation. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
From day 4 of treatment onwards an abnormal posture of the tail was noted in all Group 6 animals (1000 mg/kg); for females this was observed up to and including Day 48. Directly after dosing, the rats walked around the cage with their tails held up high and slightly curled. A veterinarian observed this reaction and it was considered to be most likely related to an unpleasant taste of the formulation. This would also correlate with the observed salivation.
Any other clinical signs noted, including hunched posture and piloerection, occurred incidentally and within the range of background findings. These were identical to those to be expected for rats of this age and strain which are housed and treated under the conditions in this study. In addition, there was no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Female body weights and body weight gains of the 1000 mg/kg groups tended to be higher than the concurrent controls. This was statistically significant for group 6 females during gestation and lactation period. However, the gestation and lactation body weights and body weight gains remained within the historical control ranges and were therefore not considered treatment related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
A slight, but statistically significant, increase in absolute and relative food consumption during gestation and lactation period was observed in Group 6, females only. This was not observed in Group 4 females, treated at the same dose level.
Overall, food consumption remained within normal limits and these findings were therefore not considered treatment related.
An isolated statistically significant difference, noted in females (higher relative food consumption at 200 mg/kg between post-coitum days 17-20), was regarded as chance finding, due to the lack of a dose-response relationship.

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

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

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

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

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

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

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

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

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested. Item-test related microscopic findings at 1000mg/kg were considered as no adverse effects.
Critical effects observed:
not specified

Analysis of dose preparations:

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

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

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

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

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

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

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

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

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

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

 

 

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
This study has been performed according to OECD and/or EC guidelines and according to GLP principles. 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: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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

Blood for toxicokinetic evaluation was sampled on treatment Day 10 (from pre-dose up to24 hours post-dose). Blood was analysed for the concentrations of the test item and a defined metabolite i.e. Ethyllinalool. A toxicokinetic evaluation was performed.

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

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

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

 

 

 

Justification for classification or non-classification

Based on the results from the key study and the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC), the substance does not need to be classified for repeated dose toxicity.