5-methylheptan-3-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15.01.2016-16.09.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-methylheptan-3-one

EC Number:

208-793-7

EC Name:

5-methylheptan-3-one

Cas Number:

541-85-5

Molecular formula:

C8H16O

IUPAC Name:

5-methylheptan-3-one

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species & Strain: Wistar rat (Rattus norvegicus)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Velaz s.r.o., The Czech Republic
Quality at delivery: SPF
Age at delivery: 6-7 weeks
Body weight at delivery: 222-296 g males, 130-172 g females
Total number of animals: 80 animals (40 males + 40 females)
Number of groups: 4 (3 treated groups and one control group)
Number of animals per group: 10 males + 10 females (all groups)
Animal identification: Individual identification of animals by tail tattoo and individual identification of boxes
Housing: The study animals were housed in Macrolon Tecniplast cages (2-3 animals of the same sex/cage) in barrier laboratory conditions at building No.2, part 1, room No. 2. Room temperature was 19-25°C and the relative humidity was between 30 70 %. The room was monitored and ventilated. Fluorescent lighting was provided illumination 12 hours per day. Feed and water containers were changed and sanitized at least once weekly. Tier Wohl Super (JRS Germany) was used as bedding, except in the metabolic cages.
Diet: During the acclimation and study periods the animals were fed with standard pellet diet of monitored quality. Feed “Myši chov” (Sehnoutek a synové v.o.s., Czech Republic) was provided ad libitum. The diet is tested twice a year for possible toxic or microbiological contamination. There were no contaminants in the diet at levels that could reasonably be expected to affect the purpose or integrity of the study. Copies of the analyses are kept in the archive of MediTox s.r.o.
Water:Water of monitored quality was supplied ad libitum during the acclimation and study periods. There were no contaminants in the water at levels that could reasonably be expected to affect the purpose or integrity of the study. Copies of the analyses are kept in the archive of MediTox s.r.o.
Acclimation: The rats were acclimated for 8-11 days. No prophylactic or therapeutic treatment was given during the acclimation or study period. Only animals in good health conditions were used for the study.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral administration as an intended route in animal is required by the corresponding international guideline. Doses were selected based on the data from 14-day Dose Range Finding study in rats (MediTox study No. 511/15).

Vehicle:

corn oil

Details on oral exposure:

The Test Item application formulation was prepared weekly as suspension in corn oil. The doses were 100 mg/kg (D1), 300 mg/kg (D2) and 1000 mg/kg (D3) respective 700 and 500 mg/kg. Test article were administered orally via oral gavage in application volume of 0.5 mL/100g body weight. The control animals were treated with vehicle in the same regime and volume as high dose group. Preparation of the suspension was performed at MediTox s.r.o.
For the preparation of a homogenous suspension the appropriate amount of TI was suspended in corn oil using magnetic stirring to enhance the rate of a homogenous suspension.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three samples (top / mid / bottom, approx. 10 ml of each) per all dose levels from application formulation in week 1 and week 13 of the study were sent to the Test Site II for analysis within 1 week after preparation. Similar samples were taken and shipped for analysis from the vehicle (vehicle only, with no added Test Item). The total of 24 samples were transferred into appropriately labelled and sealed Eppendorf tubes at ambient temperature 15-25°C until transport to the Test Site II. The Eppendorf tubes were transported at ambient temperature together with appropriate documentation (Delivery protocol). Analysis of these samples and the results obtained are to be reported in a separate Analytical Phase report, appended to the Final report. Measured concentrations of the Ethylamylketon were in the range of 92-100% of the nominal concentrations and no interfering component was detected in the control samples.

Duration of treatment / exposure:

Each animal from the dose groups was orally administered by the determined dose of the Test Items as suspension in volume of 0.5 mL/100 g b.w. every day for 90 consecutive days.
The control animals were treated with vehicle in the same regime and volume. Individual doses were adjusted according to the weekly body weight.

Frequency of treatment:

every Day for 90 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Medium dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose
Due to marked decrease of body weight and food consumption in D3 group the high dose was reduced after approval of the Sponsor to 700 mg/kg from the day 40 and again reduced to 500 mg/kg from the day 51 of the study.

Doses / concentrations 4

Dose / conc.:

700 mg/kg bw/day (nominal)

Remarks:

High dose
Due to marked decrease of body weight and food consumption in D3 group the high dose was reduced after approval of the Sponsor to 700 mg/kg from the day 40 and again reduced to 500 mg/kg from the day 51 of the study.

Doses / concentrations 5

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

High dose
Due to marked decrease of body weight and food consumption in D3 group the high dose was reduced after approval of the Sponsor to 700 mg/kg from the day 40 and again reduced to 500 mg/kg from the day 51 of the study.

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Each animal from the dose groups was orally administered by the determined dose of the Test Items as suspension in volume of 0.5 mL/100 g b.w. every day for 90 consecutive days.
The control animals were treated with vehicle in the same regime and volume. Individual doses were adjusted according to the weekly body weight.
According to the Study plan the doses administered were 100 (D1), 300 (D2), and 1000 (D3)mg/kg. Due to marked decrease of body weight and food consumption in D3 group the high dose was reduced after approval of the Sponsor to 700 mg/kg from the day 40 and again reduced to 500 mg/kg from the day 51 of the study.

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
Daily Observations: All rats were observed for clinical signs, morbidity or mortality daily in acclimation and twice a day during the administration period. Onset, duration and severity of any signs were recorded. Clinical observations included: Signs of toxicity, changes in the skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling, the presence of clonic or tonic movements and stereotypes.
Detailed Clinical Observation: Once before the first administration, and once a week thereafter, detailed clinical observation was made in all the animals. This observation was made outside the cage in a designated area and preferably at the same time of day, each time. Clinical observations were carefully recorded using a scoring system. Efforts were made to ensure that variations in the test conditions are minimal and that observations are preferably conducted by observers unaware of the treatment. Signs noted included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., self-mutilation, walking backwards) were recorded.
Functional Observation (Modified Irwin Test): Towards the end of the exposure period (week 13) modified Irwin test was performed. Parameters observed included:
I: General appearance, body position, spontaneous activity, respiration rate, tremor, twitches, bizarre behavior, convulsions, defecation.
II: Transfer arousal, locomotor activity, palpebral closure, piloerection, clap sound response, gait, pelvic elevation, tail elevation, touch escape, tail pinch, body temperature, positional passivity.
III: animals were picked up by its tail above the arena for scoring of trunk curl, limb grasping, visual placing, body tone, ear pinna reflex, corneal touch reflex, wire maneuver.
IV: animal was placed in supine restraint to score skin color, heart rate, limb tone, abdominal tone, lacrimation, pupil reflex, salivation, provoked bitting.
Body Weight: All animals were individually weighed at delivery, before the first administration, then weekly during the administration period.
Food consumption: Individual food consumption was recorded weekly during acclimation and administration period in all animals, mean weekly food consumption was calculated and provided in the tables of results.
Ophthalmoscopy: Both eyes of each animal were examined by direct and indirect ophthalmoscopy (Ophthalmoscope KEELER Professional, UK) during pre-treatment (week -1, Examination 1) and at the end of administration period (week 13, Examination 2).

Sacrifice and pathology:

CLINICAL PATHOLOGY
Blood Analyses: Blood samples for hematology and clinical chemistry were collected from all the animals before administration (week -1, Examination 1) and at the end of administration period (week 13, Examination 2). The animals were fasted for approx. 12-18 hours before blood sampling, but water was provided ad libitum. Blood samples were drawn under slight ether anesthesia from the retro-orbital venous plexus into tubes Vacuette, Greiner bio-one, containing K3EDTA (hematology) and sodium citrate (coagulation) and TAPVAL (without anti-coagulant for serum biochemistry). The blood samples for coagulation and serum chemistry were centrifuged at 4000 rpm (i.e. 2415 G) for coagulation and 6000 rpm (i.e. 3622 G) for serum chemistry for 15 minutes and transferred into plastic tubes Eppendorf. Serum for clinical chemistry was transferred into appropriately labeled and sealed Eppendorf tubes and frozen at the temperature of -20º C or below until analyses. The biochemical parameters of serum samples listed in Table 1 and 2 were determined using the Dimension Vista 1500 system.
Urinalysis: Urinalysis tests were performed before the first administration (week -1, Examination 1) and at the end of administration period (week 13, Examination 2) in all the animals. Urine samples from animals were collected in metabolic cages in interval two hours after previous oral administration of water in volume of 2 mL/100 g of b.w. Macroscopic analysis and semi-quantitative biochemical analysis (Automatic AnalyzerAution MINI, ARKRAY, Japan) were performed. The parameters listed in Table 3 were determined.
Terminal Observation
Necropsy: All animals on study survived to their scheduled necropsy, received a complete postmortem examination and a full set of tissues was collected and preserved. At the end of the administration period (Day 91), the rats were euthanized and necropsied. All the rats were weighed. Euthanasia was conducted by a cervical dislocation in deep ether anaesthesia. The postmortem examination included examination of the external surface of the body, all orifices of the body, and the cranial, thoracic, abdominal, and pelvic cavities and their contents. A full set of tissues was collected and fixed as per the list given in Table 4.
Organ Weight: The organs specified in the list above were weighed after trimming of fat and other contiguous tissues. Contra-lateral organs were weighed together. Organ weights and terminal body weights were used for the calculation of organ-to-body weight ratios.
Fixation: With the exception of the eyes, optic nerves, testes and epididymides, either whole organs or samples of the tissues listed were preserved in 4 % neutral buffered formaldehyde. The eyes, optic nerves, testes and epididymides were fixed in Davidson's fluid.

Histotechnique: Histological slides were made by common paraffin technique and stained with hematoxylin and erythrosine. Bones were decalcified with formic acid.
Histopathology: Full histopathology was carried out on the preserved organs and tissues of all animals in the control and the high dose groups.

Statistics:

Statistical Analysis:
Statistical software GraphPad Prism (version 4, last internal validation 11.08.2015) was used for statistical evaluation in this study.
Group mean and standard deviations were calculated for body weight values, food consumption, clinical pathology values (except for urinalysis) and organ weight values (absolute and relative weight values). Absolute and relative organ weight values for test article dosed groups were compared to those of control using the Student’s t Test. The data for males and females were separately analyzed to determine whether the analyzed parameters of treated groups (D1, D2, D3) are significantly different from those of controls at the 95.0 % confidence level.
The analyzed data were tested for normality (Kolmogorov–Smirnov test) and homogeneity of variance (Bartlett’s test or F test).
The data for males and females were separately analyzed using ANOVA followed by Dunnett’s Multiple Comparison Test. The F-test in the ANOVA tests whether there are any significant differences amongst the means at the 95.0 % confidence level. With small sample sizes (less than 10), the normality of the data becomes increasingly uncertain and nonparametric methods such as Kruskal–Wallis test (which compares medians instead of means) are more appropriate, so we used it too, followed by Dunn’s Multiple Comparison Test.
Statistically significant difference at the 95.0 % confidence level is printed in tables in bold. The difference between treated group (D1, D2, D3) and negative control is marked by asterisk. One asterisk (*) is used for significant difference only between means. Two asterisks (**) were used in cases for significant difference only between medians. Three asterisks (***) denote statistically significant difference between means and medians at the 95.0 % confidence level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the acclimation period, no clinical signs were recorded in any of the animals in the study.
No clinical signs of toxicity were observed during the treatment period in the animals from the control, low-dose (D1), and the middle-dose (D2) groups.
However marked clinical signs characterized by decreased motility, piloerection and salivation were observed in the high dose groups.
All males in the group D3 (1000 mg/kg) and two D3 females (F76, F78) showed repeated occurrence of piloerection, a marked decrease of motility and/or salivation. These symptoms appeared always 3-10 minutes after the administration and disappeared after 2 hours except piloerection. These clinical findings started from the day 24 of the Test Item administration period and continued after the dose reduction to 700 mg/kg from day 40 of the study. After the second reduction of the dose to 500 mg/kg (from day 51 of the study) above described clinical symptoms progressively disappeared in females by the day 56 and in males by the day 70 of the administration period.
The detailed clinical examination performed weekly did not reveal any other clinical symptoms of toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean and individual body weight values of males and females in the control, low-dose (D1), and the middle-dose (D2) groups increased during the administration period.
A marked and statistically significantly lower mean body weight (-15%) was found in males from the high dose group (D3) in week 4 of the administration period compared to controls. Body weight loss was observed thereafter until week 6 and males started to gain weight slightly in the week 7 i.e. one week after the first high dose reduction. This slight increase continued until the end of the study, but the mean body weight of D3 males stayed statistically significantly lower by approx. 20-25% than that of controls.
A statistically significant lower in the mean body weight (-11 %) was found in females from the high dose group (D3) in week 6 of administration period compared to controls. Body weight loss was observed thereafter until week 7 and females started to gain weight slightly in the week 8 i.e. two weeks after the first high dose reduction. This slight increase continued until the end of the study, but the mean body weight of D3 females stayed significantly lower by approx. 12-13% than that of controls.
The described decrease of the body weight in males and females was caused by the Test Item administered.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Several statistically significant differences of the mean food consumption were found in the D1 and D2 treated males and females during the administration period compared to control animals without affecting their body weight.
A statistically significant decrease in the food consumption by 11-28 % was found in males from the high dose group (D3) from week 4 onwards and in females from this D3 group by 9-31 % from week 1 to the end of administration period compared to the controls. There was a correlation between the reduced food consumption and the decreased body weight gain in males and females of the high dose group (D3).
The described decrease of the food consumption in rats from high dose group was caused by the Test Item administered.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related findings were obtained during ophthalmoscopic examinations.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Individual and mean values of all hematology parameters measured prior to start of the administration (Examination 1) varied within the reference limits1.
Red Blood Cells Count (RBC, HGB, HCT, MCV, MCH, MCHC), Erythroblasts Count (Ebl)
Usual inter-group variability was observed in red blood cells parameters during the study.
A few statistically significant differences as compared to control group were found in the Examination 1: Lower mean RBC, HGB and HCT values in D2 females, lower HCT value in D1 females and higher MCHC value in D1 and D2 females. In the end of the administration period (Examination 2), slight increase of erythrocytes count (RBC) as compared to the initial values was observed in all the groups (males and females) including control. All mean values varied within the reference limits. No statistically significant differences were identified between the control and dosed groups.
Corresponding changes were observed also in hemoglobin level (HGB) and hematocrit (HCT) values - slight increase in comparison to the initial values (Examination 1) which was of similar extent in all groups. In the Examination 2, statistically significantly lower mean HCT value was found in D3 females, however it was lower already in the Examination 1.
Slight decrease of MCV and MCH mean values in the Examination 2 as compared to initial values (Examination 1) was observed in all monitored groups. No statistically significant differences were identified between the control and dosed groups.
Statistically significantly higher mean MCHC value was observed in D3 females (but not in males) in the Examination 2 as compared to control group, however, this values were higher already in the Examination 1. Generally, mean MCHC values varied in all groups within the reference limits.
Normal occurrence of erythroblast (Ebl) was observed in all groups during the study.

White Blood Cells Count and Differential Leucocytes Count (WBC, SN, BN, EO, BA, LY, MO)
Slight decrease of total leucocyte counts as compared to the initial values (Examination 1) was observed in the Examination 2 in all monitored groups including control, where the range of decrease was 10 – 11 %. However, the range of decrease in D3 males and females was 22 – 27 % as compared to the Examination 1 and in D3 females the mean WBC value was even statistically significantly lower as compared to concurrent control. A trend to total leucocyte decrease could be seen in D3 males and females.
Only slight variations were observed during the study in the differential leucocyte count parameters, all mean values varied within the reference limits.
Decrease of mean absolute lymphocytes count was observed in the Examination 2 as compared to initial values in all groups (except of D2 females) which was more pronounced in D3 males and females and even statistically significant as compared to control group. Reduction in lymphocyte count was the most probably reason for reduction in total leucocyte count.
Coagulation (PLT, APTT, PT)
Mean PLT values varied within the reference limits in males and females during the study. In the Examination 2, increase of mean PLT counts were observed in all groups in males as compared to Examination 1 which was more pronounced and statistically significant (as compared to concurrent control) in D3 males. On the contrary, slight decrease or no change in comparison with the initial values was observed in female groups. No statistical significance was proved between control and dosed female groups.
Statistically significantly lower mean APTT values as compared to the control group were observed in all dosed groups of females in the Examination 1. In the Examination 2, increase of mean APTT values as compared to initial examination was observed in all monitored groups, however more marked in D3 males and females.
As far as PT is concerned, statistically significantly lower mean values as compared to control group were observed in the Examination 1 in D1 and D2 females. In the Examination 2, statistically significantly higher mean value in comparison to control group was observed in D3 females while in D2 and D3 males, statistically significantly lower mean values were observed.

Overall, hematology and coagulation parameters did not reveal any findings that could be connected with the effect of the Test Item administered except a trend to decrease of absolute lymphocyte and total leucocyte counts in D3 males and females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Generally normal variations of individual and mean values of serum glucose and serum elements (Na, K, Cl, Ca) were observed in males and females during the study. A few statistically significant differences observed between the control and dosed groups either occurred in the Examination 1 (before the start of administration) or were isolated, of minimum extent without biological significance.
In the end of the administration period (Examination 2), decrease of serum phosphorus mean values was observed in all groups in males and females as compared to initial values, which was statistically significant in D3 females as compared to concurrent control. Because of involvement in energetic balance, mild to moderate decrease is common and is of minimal biological significance.
Mean values of serum urea and creatinine concentrations varied within the reference limits in all monitored groups with some statistically differences between control and dosed groups, however without any considerable changes when compared Examination 1 and 2.
Mean serum bilirubin concentrations were within the reference limits1 in all monitored groups. In the Examination 2 an increase of mean values as compared to initial values was observed in all the groups, however statistically significant difference as compared to concurrent control was proved in D2 and D3 males and D3 females.
Slight variations within the reference limits were observed in serum bile acids concentration in all monitored groups.
Serum enzymes (LDH, AST, ALT, GMT, ALP) activities observed during the study varied mostly within the reference limits or just around the upper reference limit (ALT, Examination 2, males and females) without any considerable differences between the control and dosed groups. Increase of ALT found in the Examination 2 in all dosed groups in males and all groups including control in females as compared to the Examination 1 was of similar, biologically insignificant extent.
Slight increase of total cholesterol serum concentration was observed in the Examination 2 as compared to Examination 1 in all groups (except of female control group) which was more pronounced in D3 males and females, however no statistically significant differences were proved between the control and dosed groups. Mild, biologically insignificant increase of total cholesterol could be seen, however it should not extend the upper reference limits. All mean total cholesterol values found in the Examination 2 varied within the reference limits both in males and females.
Statistically significantly lowered mean TGC serum concentration as compared to control group was observed in D2 and D3 males (not in females), however comparing the Examination 1 and 2, the range of changes was similar in all groups.
Mean total protein values found in the Examination 1 varied within the reference limits in all groups. In the Examination 2, slight increase was observed in comparison to initial values in all monitored groups up to the upper reference limit. The extend of changes was similar in all the groups, no dose-dependence was proved.
Similar increase was observed in serum albumin and globulin concentrations in the Examination 2 as compared to Examination 1. Only isolated statistically significant differences were identified between the control and dosed groups (higher mean Alb values in D3 males, Examination 2 and higher mean Glo value in D2 females, Examination 2).
No considerable changes in Alb/Glob ratio was observed in the Examination 2 as compared to initial values. Isolated statistically significantly higher mean Alb/Glo value found in D3 males corresponds to changes found in TP, Alb and Glo in this group.

Overall, mostly normal variations of the clinical chemistry parameters were observed during the study in the monitored groups.
A trend to an increase of bilirubin at the end of the administration period could be seen in D2 and D3 males and in D3 females

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urine samples of the tested and control animals were clear and of yellow colour during the entire study.
Slight increase of volume of urine samples collected was observed in D2 and D3 males (but not in females) in the Examination 2 as compared both to Examination 1 and concurrent control.
pH values varied mostly within 6.0 – 8.5 with sporadic values of 5.5 and 9.0. Specific gravity varied within <1.005 – 1.015 kg/l in all monitored groups without any substantial differences between control and dosed groups.
Normal findings were observed in glucose and bilirubin occurrence in all monitored groups in the course of the study. Normal values of urobilinogen (< 34 µmol/l) were detected in most of the animals of all monitored groups with sporadically higher values (34 – 100 µmol/l) occurred throughout the groups and examinations. Normal values of nitrites (0 Arb. U.) was found in most of males and females from all monitored groups during the study with isolated higher values (1 Arb. U.) found independently on the dose group and examination.
Higher variations were observed in protein occurrence in all groups including control. The values mostly varied within usual ranges (< 0.1 – 0.7 g/l) with a few cases of slightly higher protein occurrence (1.0 – 2.0 g/l) independently on the dose and examination with isolated high protein occurrence (¿ 6.0 g/l) found in D2 group in the Examination 2 (1 male from 10). Generally, no substantial differences were observed between the individual groups.
No considerable differences were observed between the groups in leucocyte occurrence. In most of the animals the values varied within 25 – 500 Leu/µl independently on the group and examination.
Higher variability of ketones occurrence was observed in the animals. Basically, the values varied within usual ranges (< 1.0 – 1.0 mmol/l), however, higher values were identified in Examination 1 even in control animals (2.0 – 4.0 mmol/l, 6 males from 10 and 2.0 mmol/l, 1 female from 10).
In D1 group, usual range was observed in both examinations with isolated values of 2.0 mmol/l.
In D2 group, higher ketones occurrence (2.0 – 4.0 mmol/l) was observed in 4 males from 10 in the Examination 1 and in 7 males from 10 in the Examination 2. Usual range was observed in females in the Examination 1. Higher values (2.0 mmol/l) was observed in 1 female from 10 in the Examination 2.
In D3 group, higher ketone values (2.0 – 4.0 mmol/l) was found in 5 males from 10 in the Examination 1 and in all males in the Examination 2. In females, usual range of values (< 1.0 – 1.0 mmol/l) was observed in both examinations.

In summary, a slightly increased volume of urine samples and ketones occurrence was observed in D3 males (but not in females) at the end of the administration period as compared to both the control group and the Examination 1. No substantial differences were observed between the control and dosed groups in the other urinalysis parameters

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

No functional observation of toxicity was observed at the end of treatment period in the animals from low-dose (D1) groups compare to control group.
The animals form the high and the middle dose showed lower degree of activity when gross appearance was observed in their original housing cage.
After transfer the animals in novel environment in all animals of control, low (D1) and middle (D2) dose an immediate movement without any freeze period was observed unlike in three male and one female animals of high (D3) dose animal when brief to prolonged freeze was observed before the movement. In one male of this group, the movement was significantly reduced/limited and four male animals and three female animals showed slightly abnormal movement of the hind limbs. These findings might also relate to observed flattened pelvic elevation in two male animals of high dose group. No other differences were noted within observation of behaviour in novel environment among the groups.
During reflexes and reaction to simple stimuli assessments most of the observation of the dose groups were similar to the control group. Only in four male animals of the high dose group a slight grip on the grid during a horizontal backward pull was observed. During the wire maneuver the more difficulties to grasp the wire with hind limbs display males most probably due to higher body weight. However, four male animals of the high dose group, two male animals and one female animal of middle group and two male animals of low dose fell within a few seconds.
Only difference of measurement recorded on restrained animals was observed in limb tone where slight resistance or no resistance was mainly observed in high dose animals (four male animals and two female animals).
At grip strength, motor coordination and locomotor activity testing, the female animals of all groups held to inverse grid screen much longer in comparison to the male animals. Although the control animals on average held to the screen longer when compare means of the groups, the individual differences within each group were too large to determine difference among the groups. On the other hand the three male animals of the high dose group fell down from the grid screen during negative geotaxis test.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males:
Male rats of the D2 and D3 dose group showed significant increase in relative weight of the liver and kidneys. In the D3 males the increase in relative weight of the spleen, brain, heart, adrenals, testes and epididymides was found. On the other site, absolute organ weight of spleen, brain, heart, thymus and epididymides were lowered.

Females:
In the D3 females, decrease in absolute weight of the brain, heart and uterus was revealed. Relative liver weight of the D3 females and relative kidneys weight of the D2 and D3 females was significantly increased when compared to controls.
Described increase in relative weight of the liver and kidneys of D2 and D3 rats was dose dependent and was most probably in correlation with marked decrease of their body weight caused by the Test item administered.

In summary, organ weight analysis found treatment-related increase of the liver and kidneys relative weight in D2 and D3 rats but not for the absolute organ weights. No gross or histopathological changes in the liver and kidneys indicative of a toxic effect were observed in D3 animals and, therefore, these findings were considered to be of minor toxicological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Liver
Small foci of chronic inflammation were revealed in one control female as spontaneous finding. Small white focus was grossly found in the medial liver lobe of control male M7.

Kidneys
The lesions in the kidneys concerned control animal only (corticomedullary mineralization), or both control and administered animals (hydronephrosis) therefore they were not related to the Test item administered.
Solitary hyaline casts found in the kidneys of one D3 male and one D3 female were of spontaneous origin.

Lungs
Blood aspiration found in the lungs of some control and administered rats and venostasis revealed in the lungs of one D3 female were terminal lesions without direct relation to the substance tested.

Hematopoietic and lymphatic systems
Cervical lymph nodes of certain control and administered animals showed signs of mild to moderate reactive hyperplasia. Origin of this finding is not clear but its relation to the Test item administered could be excluded.
Mild hemorrhage revealed in the cervical lymph nodes of three control rats is a terminal lesion without direct relation to the treatment.
Mild focal hemorrhage in the thymus of two control and one D3 animal is a terminal lesion without direct relation to the treatment.

Gastrointestinal tract
No treatment-related findings were revealed in organs of the gastrointestinal tract. Origin of one small erosion revealed in the stomach mucosa of administered male M36 – D3 is unclear and its relation to the substance tested is not probable. Small mucosal cyst found in the stomach of the same rat is of spontaneous origin.

Male and female reproductive systems
Atrophy of some tubules was revealed in the testes of one D3 male as spontaneous lesion. Reproductive system of the administered males was without any treatment-related lesions.
Hydrometra observed in some control and administered females is considered to be oestrous cycle - associated change with no relation to the Test item.

In addition to organ weight changes in liver and kidneys (see subchapter organ weights), no treatment-related pathological change was noted in any other organ of D3 animals. Other lesions found in the treated animals were either of spontaneous character or they were not in direct relation to the Test item administered.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Liver
Histopathological examination revealed marked focal necrosis of liver parenchyma with early organization of unclear origin. Venostasis observed in the liver of one control male and one D3 female is terminal finding without direct relation to the treatment.
No treatment-related lesions were found in the liver parenchyma.

Hematopoietic and lymphatic systems
Microscopic examination of the spleen did not reveal any treatment-related pathological changes.

Other microscopic findings were observed in either control animals only, or in both control and administered animals, or they were of a subsidiary nature, with no relation to the substances tested.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

5-Methylheptan-3-one is not classified for specific target organ toxicity (STOT) (repeated exposure)

Executive summary:

The Test Item, Ethylamylketon, was administered orally as suspension in the doses of 100 (group D1), 300 (group D2), and 1000 (group D3) mg/kg for 90 consecutive days. The control animals were administered with the vehicle in the same regime. Due to marked decrease of body weight and food consumption in the D3 group the high dose was reduced to 700 mg/kg from the day 40 and again reduced to 500 mg/kg from the day 51 of the study. These effects were considered to be adverse.

Ethylamylketon, orally administered to rats in the high dose for 90 days, caused marked clinical signs characterized by decreased motility, piloerection and salivation. These changes were possibly related to the marked decrease of food consumption and consequently to the marked reduction of body weight of the rats. The functional observation where decreased motility, changes of movement, locomotor activity and reaction were mainly observed in high dose animals was in accordance with the clinical observations.

Relative liver and kidneys weight was increased in the D2 and D3 rats at the end of the study. A trend to increase of bilirubin level was seen in D2 and D3 males and D3 females at the end of the administration period. These findings were considered as non-adverse and of minor toxicological relevance in the absence of histopathological findings in these organs of D3 males and females.

A slightly increased volume of urine samples and ketones occurrence was observed in D3 males at the end of the administration period.

Considering the effects on body weight and food consumption in the high dose group induced by the Test Item the no observed adverse effect level (NOAEL) for Ethylamylketon was determined to be at the mid dose level for male and female animals (300 mg/kg body weight).