6-methylhept-5-en-2-one

Endpoint summary

Description of key information

Repeated dose toxicity (OECD TG 408): NOAEL = 200 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:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted guideline study conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF Aktiengesellschaft Experimentelle Toxikogie und Ökologie Ludwigshafen

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: CrlGlxBrl/Han:WI
- Source: Charles River, Germany
- Age at study initiation: 41 - 43 days
- Weight at study initiation: males mean per treatment group at day 0: 156-157 g, females mean per treatment group at day 0: 123-124 g
- Diet: ad libitum, Kliba feed,
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Olive oil was taken in a graduated flask. The appropriate amount of test substance was weighed in, filled up to the desired volume with the vehicle, and mixed using a magnetic stirrer. These solutions were prepared in intervals of no longer than 7 days and stored under N2.

VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of test substance in vehicle was determined over a period of 7 days at room temperature prior to the start of the study. As the preparations were clear solutions, no homogeneity analyses were carried out. Concentration control analyses of the test substance preparations were performed in all concentrations at the start and the end of the administration period

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Dose / conc.:

50 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)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day for evident signs of toxicity or mortality on weekdays (morning and afternoon), once at weekends (morning) and additionally daily after application of the test substance.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of the administration period and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of administration, thereafter once weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: once weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of the administration period the eyes of all animals were examined for any changes using an ophthalmoscope after administration of a mydriatic. At the end of the study, the animals of the control and high dose group were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Source: retroorbital venous plexus
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex at the end of the application period
- Parameters examined: determined in blood with EDTA-K3 as anticoagulant using a hematology analyzer: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count. Prothrombin time was determined using a ball coagulometer

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Source: retroorbital venous plexus
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex at the end of the application period
- Parameters checked in table [No.?] were examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

URINALYSIS: Yes
- Time schedule for collection of urine: in the morning
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parametersexamined: volume, color, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment. With the exception of volume, color, turbidity, sediment examination and the specific gravity, all the uine constituents were determined semiquantitatively using test strips and a reflection photometer. The specific gravity was determined using a urine refractometer. The sediment was evaluated microscopically.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: was performed towards the end of the study, starting at about 10.00 a.m.
- Dose groups that were examined: all animals
- Battery of functions tested: started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable:
- Home cage observations: The animals were observed in their closed home cages; any disturbing activities were avoided during these examinations in order not to in-fluence the behavior of the animals. Attention was paid to posture, tremor, convulsions, abnormal movements, impairment of gait and general observations.
- Open field observations: The animals were transferred to a standard arena (50x37.5 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined: behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes / pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements / stereotypics, impairment of gait, activity/arousal level, feces, urine and number of rearings.
- Sensorimotor tests / reflexes: The animals were removed from the open field and subjected to following sensorimotor or reflex tests: approach response, touch response, vision, pupillary reflex, pinna reflex, audition, coordination of movements, behavior during, vocalization, pain perception, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test
- Motor activity assessment: was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The measurements started at about 2.00 p.m. and the number of beam interrupts were counted over 12 intervals, each lasting 5 minutes. The period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack. Measurements ended exactly 60 minutes thereafter.

SPERM PARAMETERS: Yes
- Time schedule: immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.
- Parameters examined: sperm motility, sperm morphology, sperm head count (cauda epididymis), sperm head count (testis)

ESTRUS CYCLE DETERMINATION: Yes
- Time schedule: Vaginal smears for cycle determination were prepared in the morning and evaluated from day 63 until day 91 of the study.
- The differentiation was conducted to following cycle stage evaluated by appearance in vaginal smear:Diestrous (Leucocytes, few nucleated, epithelial cells); Proestrous (Single leucocytes, many nucleated and few horny epithelial cells); Estrous (only horny epithelial cells); Metestrous (Leucocytes, some horny epithelial cells and some nucleated epithelial cells)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
- animals were sacrificed by decapitation under CO2 anesthesia, exsanguinated animals were necropsied and assessed by gross pathology.
- Organ weights from all animals sacrificed at scheduled dates were determined: Anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart, thymus, prostate gland

HISTOPATHOLOGY: Yes
- following organs were fixed in 4% formaldehyde solution, histopathologically processed and examined by light microscopy: All gross lesions, salivary glands (mandibular and sublingualis), esophagus, stomach (forestomach and glandular stomach), duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord (cervical, thoracic and lumbar cord), eyes, adrenal glands, thyroid glands, parathyroid glands, trachea, lungs, pharynx, larynx, nose (nasal cavities), aorta, heart, bone marrow (femur), lymph nodes (mandibular and mesenteric), spleen, thymus, kidneys, urinary bladder, ovaries, oviducts/uterus/vagina, prostate gland, seminal vesicles, female mammary gland, skin, skeletal muscle, sternum with marrow, femur with knee joint, extraorbital lacrimal glands
- The left testis and the left epididymides were fixed in BOUIN's solution and embedded in paraplast.
- In the kidneys immunohistochemical staining and staining according to Mallory Heidenhain for alpha-2u-globulin detection was performed

Statistics:

Means and standard deviations of each test group were calculated for several parameters. Further statistical analyses were performed:
- Dunnett test: Food consumption, body weight, body weight change, food efficiency, mean estrous stages
- Kruskall-Wallis test: Feces, rearing, grip strength length forelimbs, grip strength length hindlimbs, landing foot-splay test, motor activity, clinical pathology except differential blood count, pathological weight parameters (if p-value < = 0.05 Wilcoxon test was additionally performed)
- Fishers exact test: Urinalysis except volume, color, turbidity and specific gravity; abnormal sperm > 4%
- Wilcoxon test Total spermatids/g testis, total sperm/g cauda epi., % motility; a2µ-globulin

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All animals of the high dose group showed slight to moderate salivation on several days from day 8 until the end of the study.
- Other treatment related findings were not observed.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Body weight in male animals of the high dose group was throughout the whole study period decreased with a maximum of -7.2% on study day 91.
- The body weight change of these high dosed males was also decreased. Although these effects were not statistically significant, the impairment of body weight as well as body weight change in male animals of the high dose group was assessed as compound-related.
- Body weight in females of the high dose group was statistically significantly decreased (-6.7%) on day 63, only.
- Body weight change in females of this dose group was statistically significantly decreased up to -16.4% from day 35 to day 84, with exception of day 70. These findings were also assessed as being related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Food consumption in females of the high dose group was statistically significantly decreased (up to -13%) from day 28 to day 49. This finding was assessed as being related to treatment.
- In the other dose groups no treatment dependent changes were observed.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

- Significantly decreased in males of the high dose group on days 21, 35, 63, 77 of the study compared to controls. This was assessed as being related to treatment.

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

HAEMATOLOGY
- At the end of the administration period increased platelets were found in the peripheral blood of the high dose animals of either sex. Platelet counts were also higher in the mid dose females.
- No other hematology examinations did not reveal treatment-related changes.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- After 4 weeks of test substance administration aspartate aminotransferase activities were decreased in the high dose animals of either sex.
- A slight increase in alkaline phosphatase activity was seen in the high dose males.
- No treatment-related changes were observed in other serum enzyme examinations.
- Increased calcium, total protein, albumin and cholesterol concentrations in the high dose animals of both sexes.
- Increased calcium, total protein, albumin and cholesterol values were found in the mid dose males
- In the sera of the high dose females chloride concentrations were decreased and inorganic phosphate, urea, total bilirubin, globulins and magnesium levels were increased.
- No test substance-related changes were noted in the other blood chemistry examinations.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

URINALYSIS
- Urine samples of 5 out of 10 high dose males appeared cloudy and the reagent strip test indicated increased blood in the urine specimens of the high dose males.
- Microscopic examination of the urine sediments of the high dose males revealed increased numbers of degenerated renal tubular epithelial cells and degenerated transitional epithelial cells as well as granular casts and epithelial cell casts at the end of the study.
- The test compound did not affect the other urine parameters.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver - significantly increased in males (+29.6%) and in females (+21.9%) of the high dose group.

Kidney - significantly increased in males of the high (+28.0%), mid (+16.5%) and low dose groups (+14.3%) in a dose-related fashion and in females of the high dose group (+14.3%).

Treatment related changes in mean relative organ weights (related to terminal body weight):
Liver - significantly increased in males (+40.7%) and in females (+29.7%) of the high dose group.
Kidney - significantly increased in males of the high (+38.7%), mid (+16.3%) and low dose groups (+11.6%) in a dose-related fashion and in females of the high dose group (+23.4%). The mean absolute weight of the epididymides was incidentally although significantly increased in males of the low dose group (+8.1%).

Not considered treatment related:
- no dose-response relationship: slight, although not significant increase of the mean terminal body weight (+3.0%), no such observation in the more reliable relative weight observed
- no morphologic correlate; The mean weights of the adrenal glands were significantly increased in males (+17.6%) and in females (+15.2%) of the high dose group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Not considered treatment related:
-a slight, although not significant decrease of the mean terminal body weight in males (-7.9%) and in females (-6.2%)
-no morphologic correlate for the weight increase in this organ in either sex.

Gross lesions:
Gross lesions were noted in the epididymides and testes (organ size reduced in three high dose males ), thyroid glands (organ size reduced) and vagina (inflammation and malformation in 1 high dose female ). With the exception of reduced organ sizes of testes and epididymides in high dose males, they were either single observations or they were biologically equally distributed over the control and treatment groups with no obvious relationship to treatment.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Functional observational battery and motor activity measurement
- All findings were assessed as being incidental, as they occurred in single animals, only, or were equally distributed between treated groups and controls.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

HISTOPATHOLOGY: NON-NEOPLASTIC
- Kidney: Increased accumulation of alpha2u globulin in the renal cortex of all male rats of the high/ mid dose group (assessed from slides stained with Mallory-Heidenhain). Increased accumulation of alpha2u globulin in the renal cortex of male rats of the low dose group (assessed from slides immunostained for alpha2u globulin).Multifocal dilation (slight or moderate) of renal tubular lumina in 7 males (high dose) or 2 males (mid dose).
- Liver:Centrolobular hypertrophy of liver cells in almost all males and in all females (high dose)
- Testis/Epididymis:Diffuse tubular atrophy in the testes of three rats and focal tubular atrophy in two other rats (high dose)Aspermia and debris in the lumen of the epididymides of three rats (high dose).

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

SPERM ANALYSIS
- In 3 out of 10 male animals of the high dose group no spermatids per gram testis and a significant reduction in the number of spermatozoa per gram cauda epididymis were observed. In these animals, a significant increase in sperm with abnormal morphology was seen and the sperm motility could not be evaluated due to insufficient number of motile sperm

ESTROUS CYCLE DETERMINATION
- No substance-related effects were obtained.

Details on results:

CLINICAL EXAMINATIONS
- Mortality: No animal died during the administration period.
- Clinical signs: All animals of the high dose group showed slight to moderate salivation on several days from day 8 until the end of the study.
- Other treatment related findings were not observed.

FOOD CONSUMPTION
- Food consumption in females of the high dose group was statistically significantly decreased (up to -13%) from day 28 to day 49. This finding was assessed as being related to treatment.
- In the other dose groups no treatment dependent changes were observed.

BODY WEIGHT AND WEIGHT GAIN
- Body weight in male animals of the high dose group was throughout the whole study period decreased with a maximum of -7.2% on study day 91.
- The body weight change of these high dosed males was also decreased. Although these effects were not statistically significant, the impairment of body weight as well as body weight change in male animals of the high dose group was assessed as compound-related.
- Body weight in females of the high dose group was statistically significantly decreased (-6.7%) on day 63, only. Body weight change in females of this dose group was statistically significantly decreased up to -16.4% from day 35 to day 84, with exception of day 70. These findings were also assessed as being related to treatment.

FOOD EFFICIENCY
- significantly decreased in males of the high dose group on days 21, 35, 63, 77 of the study compared to controls.
- This was assessed as being related to treatment.

OPHTHALMOSCOPIC EXAMINATION
- No substance-related effects were obtained.

NEUROBEHAVIOUR
- Functional observational battery and motor activity measurement
- All findings were assessed as being incidental, as they occurred in single animals, only, or were equally distributed between treated groups and controls.

ESTROUS CYCLE DETERMINATION
- No substance-related effects were obtained.

CLINICAL CHEMISTRY
- After 4 weeks of test substance administration aspartate aminotransferase activities were decreased in the high dose animals of either sex.
- A slight increase in alkaline phosphatase activity was also seen in the high dose males.
- No treatment-related changes were observed in the other serum enzyme examinations.
- Increased calcium, total protein, albumin and cholesterol concentrations in the high dose animals of both sexes.
- Increased calcium, total protein, albumin and cholesterol values were also found in the mid dose males
- Furthermore, in the sera of the high dose females chloride concentrations were decreased and inorganic phosphate, urea, total bilirubin, globulins and magnesium levels were increased.
- No test substance-related changes were noted in the other blood chemistry examinations.

HAEMATOLOGY- At the end of the administration period increased platelets were found in the peripheral blood of the high dose animals of either sex. Platelet counts were also higher in the mid dose females. - The other hematology examinations did not reveal treatment-related changes.

URINALYSIS
- Urine samples of 5 out of 10 high dose males appeared cloudy and the reagent strip test indicated increased blood in the urine specimens of the high dose males.
- Microscopic examination of the urine sediments of the high dose males revealed increased numbers of degenerated renal tubular epithelial cells and degenerated transitional epithelial cells as well as granular casts and epithelial cell casts at the end of the study.
- The test compound did not affect the other urine parameters.

SPERM ANALYSIS
- In 3 out of 10 male animals of the high dose group no spermatids per gram testis and a significant reduction in the number of spermatozoa per gram cauda epididymis were observed.
- In these animals, a significant increase in sperm with abnormal morphology was seen and the sperm motility could not be evaluated due to insufficient number of motile sperm.

PATHOLOGY
Treatment related changes in mean absolute organ weights:
Liver - significantly increased in males (+29.6%) and in females (+21.9%) of the high dose group.
Kidney - significantly increased in males of the high (+28.0%), mid (+16.5%) and low dose groups (+14.3%) in a dose-related fashion and in females of the high dose group (+14.3%). Treatment related changes in mean relative organ weights (related to terminal body weight):
Liver - significantly increased in males (+40.7%) and in females (+29.7%) of the high dose group.
Kidney - significantly increased in males of the high (+38.7%), mid (+16.3%) and low dose groups (+11.6%) in a dose-related fashion and in females of the high dose group (+23.4%). The mean absolute weight of the epididymides was incidentally although significantly increased in males of the low dose group (+8.1%).

Not considered treatment related:
- no dose-response relationship
- slight, although not significant increase of the mean terminal body weight (+3.0%)
- no such observation in the more reliable relative weight observed
- no morphologic correlate;The mean weights of the adrenal glands were significantly increased in males (+17 .6%) and in females (+15.2%) of the high dose group. Not considered treatment related:
- a slight, although not significant decrease of the mean terminal body weight in males (-7.9%) and in females (-6.2%)
- no morphologic correlate for the weight increase in this organ in either sex.

Gross lesions: Gross lesions were noted in the epididymides and testes (organ size reduced in three high dose males ), thyroid glands (organ size reduced) and vagina (inflammation and malformation in 1 high dose female ). With the exception of reduced organ sizes of testes and epididymides in high dose males, they were either single observations or they were biologically equally distributed over the control and treatment groups with no obvious relationship to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Kidney:Increased accumulation of alpha2u globulin in the renal cortex of all male rats of the high/ mid dose group (assessed from slides stained with Mallory-Heidenhain). Increased accumulation of alpha2u globulin in the renal cortex of male rats of the low dose group (assessed from slides immunostained for alpha2u globulin).Multifocal dilation (slight or moderate) of renal tubular lumina in 7 males (high dose) or 2 males (mid dose).
Liver:Centrolobular hypertrophy of liver cells in almost all males and in all females (high dose)Testis/Epididymis:Diffuse tubular atrophy in the testes of three rats and focal tubular atrophy in two other rats (high dose)Aspermia and debris in the lumen of the epididymides of three rats (high dose).

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

other: reproductive system

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (actual dose received)

System:

other: renal system

Organ:

kidney

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

no

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

Conclusions:

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) was reported to be 50 mg/kg bw/day. However, upon reviewing the study report, the toxicology expert panel concluded that the adversity of the observed effects in the mid dose group (200 mg/kg bw) is questionable. The increase in blood platelets at the female rat is minimal and could not be linked to any disease process. Furthermore, the calcium, protein, albumin and cholesterol increases observed in male rats are minimal, and did not lead to any histopathological or degenerative effects. Therefore, the review panel considers the observed effects at methylheptenone exposure levels of 200 mg/kg bw as adaptive and “non-adverse”, and will therefore use a NOAEL of 200 mg/kg bw/day for classification and human risk assessment. Based on a NOAEL of 200 mg/kg bw and the criteria outlined in Annex I of Regulation (EC) No. 1272/2008, Methylheptenone does not need to be classified for oral repeated dose toxicity.

Executive summary:

This report presents the findings of a 90-day oral toxicity study designed to evaluate the toxicity of  in 10 male and 10 female rats when administered Methylheptenone in olive oil, daily by gavage to achieve dosage levels of 50, 200, and 1000 mg/kg of body weight per day. A control group received only the vehicle. Criteria evaluation for signs of compound effect included survival, clinical observations, body weights, food consumption, food efficiency, opthalmoscopy, functional observational battery, motor activity assessment, estrous cylce determination, clinical pathology, gross pathology, organ weights, histopathology, hematology, clinical chemistry, urinalysis and sperm parameters.

 

During clinical examinations all male as well as female animals of the high dose group showed slight to moderate salivation on several study days, being clearly related to the treatment but rather caused by the physical properties of the test compound than reflecting a toxicologically relevant effect. Decreased food consumption in females, the impairment of body weight and body weight change in both sexes and the decreased food efficiency in male animals were assessed as substance-related signs of general toxicity. Regarding hematology only platelet counts were significantly increased in the high dose animals of either sex and in the mid dose females, being considered as treatment related. Since no corresponding changes were seen in the other clinical pathology examinations, this isolated finding is difficult to interpret in its original cause.

 

Reduced aspartate aminotransferase activities in the high dose animals of both sexes and slightly increased alkaline phosphatase activities in the high dose males, are considered to be test substance-related.

Since no adverse effects were observed in this study which could be associated with the fall in aspartate aminotransferase activities, the finding is considered to be of no toxicological importance. The slight increase in alkaline phosphatase activities in the serum of the high dose males is regarded not to represent any toxicologically relevant change, since changes in serum alkaline phosphatase activities may be explained by the dependence of the enzyme on the nutritional state of the animals.

Changes were also seen in various blood chemistry parameters of the high dose animals and mid dose males. These changes, eg. increases in inorganic phosphate, calcium, urea, bilirubin, total protein, albumin, globulins, cholesterol and magnesium and decreases in chloride, are regarded to be treatment-related. However, these findings could not be assigned to a specific disease.

Urine analysis revealed increased blood in the urine specimens of the high dose males and in 5 out of 10 high dose males the urine samples appeared cloudy. Moreover, in the urine sediments of the high dose males increased numbers of degenerated renal tubular epithelial cells and transitional epithelial cells as well as granular and epithelial cell casts were detected, being the result of increased desquamation of tubular cells and increased excretion of mucoprotein. These findings are suggestive of renal tubular epithelial damage in the high dose males.

Sperm analysis revealed reduced number of spermalozoa in the cauda epididymis, decreased number of homogenization-resistant spermatids in the testis and increases in the percentage of morphologically abnormal sperms in 3 out of 10 male animals of the high dose group. These results suggest that the test compound caused testicular toxicity affecting spermatogenesis at a dose level of 1000 mg/kg bw/day.

 

Concerning pathology, treatment-related weight changes and microscopic findings were noted in the kidneys (significantly increased mean absolute and relative weights and diffuse accumulation of a2u globulin in the epithelia and tubular lumina of the proximal tubules of the renal cortex and multifocal cystic dilation of renal tubules of male rats), in the liver (significantly increased mean absolute and relative liver weights (males and females) in the high dose group as well as centrolobular liver cell hypertrophy in males and females of the high dose group) and in testes (focal and diffuse tubular atrophy) and epididymides (aspermia and debris in lumen). Alpha2u globulin accumulation is a rat-specific phenomenon, and has no toxicological impact on the human situation. Although no morphologic correlate was obtained for the significantly increased absolute and relative kidney weights in high dose females, and although alpha2u globulin accumulation was not the cause of this weight change as this protein does not occur in female rats, the weight increase was regarded to be treatment-related. Perhaps the weight increase was related to an increased metabolic activity of the renal cells associated with metabolism and/or excretion of the compound via the urine.

 

Liver cell hypertrophy is coincident with increased mean absolute and/or relative liver weights, being indicative for an adaptive process, i.e. enzyme induction. Treatment-related weight changes and microscopic findings were noted in testes (focal and diffuse tubular atrophy) and epididymides (aspermia and debris in lumen).  Although testicular atrophy was only noted in five animals of the high dose group and although in two cases the atrophy was only focal with an only minimal or slight degree of severity, this lesions was regarded treatment-related, as no such findings were recorded in animals of the control group (and in animals of low or mid dose groups). No test substance related effects were observed on other male or female reproductive organs.

 

In conclusion, the administration of methylheptenon by gavage for 13 weeks caused substance-related effects as above mentioned. The target organs were kidney, liver and testes. Increased accumulation of alpha-2-µ globulin in the renal cortex is known to be a specific male rat phenomenon that does not occur in other species, including humans. The increased kidney weights in the low dose male rats were also attributed to the accumulation of alpha-2-µ globulin and are therefore also considered to be male rat specific. Thus, under the conditions of this study the no observed adverse effect level (NOAEL) was 50 mg/kg bw/day in female rats and lower than 50 mg/kg bw/day in male rats. Taking into account that the findings at 50 mg/kg bw/day were male rat specific, The author concluded in this study that the NOAEL in this study for the purpose of human risk assessment is 50 mg/kg bw/day for both sexes.

 

However, upon reviewing the study report, the toxicology expert panel concluded that the adversity of the observed effects is questionable. The increase in blood platelets at the female rat mid dose group (200 mg/kg bw) is minimal and could not be linked to any disease process. Furthermore, the calcium, protein, albumin and cholesterol increases observed in male rats are minimal, and did not lead to any histopathological or degenerative effects. Therefore, the review panel considers the observed effectsat methylheptenone exposure levels of 200 mg/kg bw as adaptive and “non-adverse”, and will therefore use a NOAEL of 200 mg/kg bw/day for classification and human risk assessment.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is a reliable guideline study performed under GLP conditions

System:

other: Renal, hepatobillary and male reproductive system

Organ:

kidney

liver

testes

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

This report presents the findings of a 90-day oral toxicity study designed to evaluate the toxicity of  in 10 male and 10 female rats when administered Methylheptenone in olive oil, daily by gavage to achieve dosage levels of 50, 200, and 1000 mg/kg of body weight per day. A control group received only the vehicle. Criteria evaluation for signs of compound effect included survival, clinical observations, body weights, food consumption, food efficiency, opthalmoscopy, functional observational battery, motor activity assessment, estrous cylce determination, clinical pathology, gross pathology, organ weights, histopathology, hematology, clinical chemistry, urinalysis and sperm parameters.

 

During clinical examinations all male as well as female animals of the high dose group showed slight to moderate salivation on several study days, being clearly related to the treatment but rather caused by the physical properties of the test compound than reflecting a toxicologically relevant effect. Decreased food consumption in females, the impairment of body weight and body weight change in both sexes and the decreased food efficiency in male animals were assessed as substance-related signs of general toxicity. Regarding hematology only platelet counts were significantly increased in the high dose animals of either sex and in the mid dose females, being considered as treatment related. Since no corresponding changes were seen in the other clinical pathology examinations, this isolated finding is difficult to interpret in its original cause.

 

Reduced aspartate aminotransferase activities in the high dose animals of both sexes and slightly increased alkaline phosphatase activities in the high dose males, are considered to be test substance-related.

Since no adverse effects were observed in this study which could be associated with the fall in aspartate aminotransferase activities, the finding is considered to be of no toxicological importance. The slight increase in alkaline phosphatase activities in the serum of the high dose males is regarded not to represent any toxicologically relevant change, since changes in serum alkaline phosphatase activities may be explained by the dependence of the enzyme on the nutritional state of the animals.

Changes were also seen in various blood chemistry parameters of the high dose animals and mid dose males. These changes, eg. increases in inorganic phosphate, calcium, urea, bilirubin, total protein, albumin, globulins, cholesterol and magnesium and decreases in chloride, are regarded to be treatment-related. However, these findings could not be assigned to a specific disease.

Urine analysis revealed increased blood in the urine specimens of the high dose males and in 5 out of 10 high dose males the urine samples appeared cloudy. Moreover, in the urine sediments of the high dose males increased numbers of degenerated renal tubular epithelial cells and transitional epithelial cells as well as granular and epithelial cell casts were detected, being the result of increased desquamation of tubular cells and increased excretion of mucoprotein. These findings are suggestive of renal tubular epithelial damage in the high dose males.

Sperm analysis revealed reduced number of spermalozoa in the cauda epididymis, decreased number of homogenization-resistant spermatids in the testis and increases in the percentage of morphologically abnormal sperms in 3 out of 10 male animals of the high dose group. These results suggest that the test compound caused testicular toxicity affecting spermatogenesis at a dose level of 1000 mg/kg bw/day.

 

Concerning pathology, treatment-related weight changes and microscopic findings were noted in the kidneys (significantly increased mean absolute and relative weights and diffuse accumulation of a2u globulin in the epithelia and tubular lumina of the proximal tubules of the renal cortex and multifocal cystic dilation of renal tubules of male rats), in the liver (significantly increased mean absolute and relative liver weights (males and females) in the high dose group as well as centrolobular liver cell hypertrophy in males and females of the high dose group) and in testes (focal and diffuse tubular atrophy) and epididymides (aspermia and debris in lumen). Alpha2u globulin accumulation is a rat-specific phenomenon, and has no toxicological impact on the human situation. Although no morphologic correlate was obtained for the significantly increased absolute and relative kidney weights in high dose females, and although alpha2u globulin accumulation was not the cause of this weight change as this protein does not occur in female rats, the weight increase was regarded to be treatment-related. Perhaps the weight increase was related to an increased metabolic activity of the renal cells associated with metabolism and/or excretion of the compound via the urine.

 

Liver cell hypertrophy is coincident with increased mean absolute and/or relative liver weights, being indicative for an adaptive process, i.e. enzyme induction. Treatment-related weight changes and microscopic findings were noted in testes (focal and diffuse tubular atrophy) and epididymides (aspermia and debris in lumen).  Although testicular atrophy was only noted in five animals of the high dose group and although in two cases the atrophy was only focal with an only minimal or slight degree of severity, this lesions was regarded treatment-related, as no such findings were recorded in animals of the control group (and in animals of low or mid dose groups). No test substance related effects were observed on other male or female reproductive organs.

 

In conclusion, the administration of methylheptenon by gavage for 13 weeks caused substance-related effects as above mentioned. The target organs were kidney, liver and testes. Increased accumulation of alpha-2-µ globulin in the renal cortex is known to be a specific male rat phenomenon that does not occur in other species, including humans. The increased kidney weights in the low dose male rats were also attributed to the accumulation of alpha-2-µ globulin and are therefore also considered to be male rat specific. Thus, under the conditions of this study the no observed adverse effect level (NOAEL) was 50 mg/kg bw/day in female rats and lower than 50 mg/kg bw/day in male rats. Taking into account that the findings at 50 mg/kg bw/day were male rat specific, The author concluded in this study that the NOAEL in this study for the purpose of human risk assessment is 50 mg/kg bw/day for both sexes.

 

However, upon reviewing the study report, the toxicology expert panel concluded that the adversity of the observed effects is questionable. The increase in blood platelets at the female rat mid dose group (200 mg/kg bw) is minimal and could not be linked to any disease process. Furthermore, the calcium, protein, albumin and cholesterol increases observed in male rats are minimal, and did not lead to any histopathological or degenerative effects. Therefore, the review panel considers the observed effectsat methylheptenone exposure levels of 200 mg/kg bw as adaptive and “non-adverse”, and will therefore use a NOAEL of 200 mg/kg bw/day for classification and human risk assessment.

Justification for classification or non-classification

Based on the available data and the review of the toxicology expert panel, the No Observed Adverse Effect Level (NOAEL) was established to be 200 mg/kg bw/day. Based on this NOAEL and  the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC), Methylheptenone does not need to be classified for repeated dose toxicity.